RU2846033C1 - Method for optically controlled theranostics using mps-cytoblockade - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics, namely to a method for optically controlled theranostics. Method for optically controlled theranostics, which includes the following steps: i) selecting a luciferase enzyme and a substrate thereof, ii) selecting a therapeutic molecule, iii) selecting nanoparticles capable of sorbing said substrate and said therapeutic molecule, iv) mixing and incubating said nanoparticles with a solution of said substrate of said luciferase for sorption of substrate into a nanoparticle, v) mixing and incubating said nanoparticles with a solution of said therapeutic molecule for its sorption into a nanoparticle, vi) conjugating said nanoparticles with said luciferase enzyme, vii) washing off said substrate which is not bound to said nanoparticle and obtaining theranostic autonomous bioluminescent nanoparticles, viii) inducing cytoblockade of the macrophage phagocytic system in an organism by administering antibodies against erythrocytes of said organism, ix) administering said theranostic autonomous bioluminescent nanoparticles into the body, x) detecting a bioluminescent signal from said autonomous-bioluminescent nanoparticles for optical monitoring of the localization of delivery of said nanoparticles carrying said therapeutic molecule to the body.

EFFECT: invention allows to perform optically controlled theranostics using nanomarkers under the action of MPS-cytoblockade, which emit light without requiring excitation with light, due to which their registration is not limited to autofluorescence of samples, including biological tissues, and also do not require additional introduction to the sample of substrates in high concentrations for minimal invasiveness and harm to the body, while having a sufficiently long-term independent bioluminescence.

10 cl, 9 dwg, 26 ex

Description

Field of technology to which the invention relates

The invention relates to the field of medical theranostics using MFS-cytoblockade technology, more precisely, to the field combining diagnostics and therapy in one drug due to the use of multifunctional nanoparticles, as well as methods for increasing the effectiveness of nanopreparations in the body due to the effect on the macrophage system of the body. Such technologies are useful for the diagnosis and therapy of diseases, conducting various studies of the state of the body.

Description of the Prior Art

The present invention relates to autonomous bioluminescent particles on which both luciferase and its substrate are located, providing conditions for their interaction and, as a consequence, the occurrence of light emission (bioluminescence). In particular, the invention is related to nanoparticles on which components of the luciferase/substrate system are located, which allows bioluminescence to be realized without external energy sources or stimuli.

Until now, there have been various approaches in the field of bioluminescence to use luciferase and its substrate to create luminous particles, but most of these solutions face problems related to the integration of these components, their stability and efficiency. The key limitations of known solutions are:

1. Separate use of luciferase and substrate: in existing approaches, luciferase and its substrate were usually introduced into the sample separately: either in solution or on different particles, which required additional mechanisms for delivering these components to the reaction site, controlling their dosing in optimal concentrations, and could also potentially lead to side reactions of excess substrate with surrounding objects, in particular with biomolecules and cells. This significantly reduces both the efficiency of using such an agent and the safety of its implementation.

2. Low stability of bioluminescent systems: Many of the existing technologies require the use of luciferase and substrate in a liquid medium, which leads to a decrease in the stability of these systems in the long term, for example, due to a reaction with an aggressive physiological environment of the agent. This limits their use in practical applications that require stable operation over a long period of time or in unstable conditions.

3. Limited ability to control light intensity: In most existing systems, luciferase and substrate interact under uncontrolled conditions, making it difficult to precisely control the intensity of emitted light. Many applications require the ability to precisely control light emission, which is not always possible using traditional technologies.

4. Use of chemical catalysts and external energy sources: In some cases, chemical catalysts or external energy sources (such as ultraviolet radiation or electrons) are required to activate bioluminescence. This significantly complicates the technology and reduces its applicability in autonomous systems.

5. Substrate delivery and release mechanisms: For luciferase to function effectively, precise control of substrate delivery is necessary, which brings additional challenges, especially in the case of in vivo use. In some systems, substrate use is not optimal due to difficulties in releasing it at the right time or due to insufficient availability to luciferase, in particular, this is due to the low solubility of many substrates, rapid excretion of the molecular substrate by the kidneys before it reaches the luciferase label in significant concentration in the body, cross-reactivity of the substrate, undesirable biological activity of the substrate.

The authors of the invention discovered an interesting phenomenon, that for nanoparticles capable of sorbing and desorbing the substrate, it is possible to create autonomous bioluminescent particles (ABLP), in which luciferase and its substrate are integrated on one particle, i.e. in which the substrate is in the pores of the particle, and the luciferase enzyme is immobilized on the surface. At the same time, autonomous bioluminescence consists in the fact that even after the removal of substrate molecules unbound to the particle, high luminescent activity of the particles can be observed due to the dosed release of the substrate and its controlled interaction with the immobilized enzyme.

This allows:

1) Increase the efficiency of the reaction due to the fact that the components are in close proximity to each other, which reduces the likelihood of their separation or insufficient reaction.

2) Ensure higher stability of the bioluminescent system by placing the components on a nanoparticle, which helps protect them from external factors and can ensure longer-term storage.

3) Control the light intensity by varying the particle size, the type of materials used, or the concentration of components, which opens up new possibilities for creating adjustable bioluminescent sources.

4) Elimination of the need for catalysts and external energy sources. The invention allows the creation of autonomous systems operating on the basis of natural chemical interaction between luciferase and its substrate, which significantly simplifies the design and increases the flexibility of use.

5) Significantly reduce the dose of substrate introduced into the body or sample.

A similar method is known from the publication of Nikitin, M.P. et al. Nat Biomed Eng 4, 717–731 (2020) https://doi.org/10.1038/s41551-020-0581-2, in which a magnetic liposome loaded with doxorubicin is administered 12 hours after the induction of MFS cytoblockade into a mouse with a tumor to which a magnet is attached. Then, the effective delivery of the liposomal drug to the tumor is visualized and confirmed using MRI, and then a significant decrease in tumor growth compared to the control, as well as an increase in mouse survival, are observed. The disadvantage of the known method is the requirement for an expensive MRI setup for detecting nanoparticles, as well as a long MRI scanning time.

From the publication El-Sayed et al. Nano Letters 2013 13 (4), 1393-1398 DOI: 10.1021/nl304123u the closest method is known, in which carbon nanotubes loaded with doxorubicin and conjugated with Luciola cruciata luciferase were introduced into an animal. Then the luciferase substrate was separately introduced into the animal. Then the distribution of nanoparticles and their accumulation in different organs were optically visualized. The disadvantage of the closest method is the need to introduce fairly high doses of the substrate, which can lead to significant toxicity, as well as generate significant autobioluminescence due to enzymatic-free photon emission. Such a step reduces the diagnostic accuracy of the data obtained.

Thus, the required technical result consists in the implementation of the method of optically controlled theranostics using nanomarkers under the action of MFS-cytoblockade, which emit light without requiring excitation by light, due to which their registration is not limited to autofluorescence of samples, including biological tissues, and also does not require additional introduction of chemical molecules into the sample (substrates, including those sorbed in other particles) or in high concentrations for minimal invasiveness and harm to the sample, while they have a sufficiently long-term autonomous (from additional chemical substrates) bioluminescence.

Summary of the invention

The aim of the present invention was to develop, based on the discovered phenomenon of autonomous bioluminescence of luciferases immobilized on nanoparticles due to substrate molecules desorbed from the nanoparticles, a method for optically controlled theranostics.

To solve the above and other goals that are obvious to a qualified specialist, the following method was proposed:

A method of optically controlled theranostics that includes the following steps:

i) select the luciferase enzyme and its substrate,

ii) select a therapeutic molecule,

iii) selecting nanoparticles capable of sorbing said substrate and said therapeutic molecule,

iv) mixing and incubating said nanoparticles with a solution of said substrate of said luciferase to sorb the substrate into the nanoparticle,

v) mixing and incubating said nanoparticles with a solution of said therapeutic molecule for its sorption into the nanoparticle,

vi) conjugating said nanoparticles with said luciferase enzyme,

vii) washing off said substrate that is not bound to said nanoparticle and obtaining theranostic autonomously bioluminescent nanoparticles,

viii) induce cytoblockade of the macrophage phagocytic system in the body by introducing antibodies against the erythrocytes of the said organism,

ix) introduce into the body the mentioned theranostic autonomously bioluminescent nanoparticles,

x) detecting a bioluminescent signal from said autonomously bioluminescent nanoparticles to optically monitor the localization of delivery of said nanoparticles carrying said therapeutic molecule in the body.

Dictionary

Unless otherwise defined, the following terms have the following meanings when interpreting the claims and description of the invention:

0) Within the framework of this description, the terms "nanoparticle", "nanoagent", "microparticle", "nanomarker", "nanotag" and their derivatives may be used interchangeably. Within the framework of this description, where applicable, the terms "bioluminescence", "luminescence" and their derivatives may be used interchangeably.

1) "immunochromatographic test" - placing a sample, which may contain a target nucleotide sequence, on a test strip made of chromatographic material, followed by lateral movement of the sample along the test strip due to capillary forces and its binding to various reagents placed on the test strip.

2) "test strip" - a chromatographic medium in which the analysis can be carried out. The test strip contains an "application zone" for applying a sample and a "binding zone" (test line) containing an immobilized binding reagent or a binding nucleotide sequence capable of binding and immobilizing the nucleotide sequence to be detected. The binding zone, as a rule, has a significantly smaller surface area than the test strip and contains an immobilized binding reagent or an immobilized binding nucleotide sequence. The binding zone can have the form of a point, line, curve, strip, or a pattern of points, lines, curves, stripes, or a combination thereof. As a rule, the direction of movement of the sample along the test strip intersects the binding zone. For the present invention, it is preferable to form a signal in the binding zone in the form of a clearly defined pattern, sharply different from the signal formed in other parts of the test strip. For example, the binding zone may be in the form of an abbreviation of the name or names of the analyte or analytes in the sample being tested.

3) The term "binding partner" refers to a pair of molecules and/or particles that are able to recognize each other (in whole or in part) and form both covalent and non-covalent bonds. There are a number of commonly used binding pairs and structures based on such pairs. Among them are systems based on the interaction of antigen/antibody, hapten/antihapten, dioxygenin/antidioxygenin F(ab')2, folic acid/folate-binding protein, biotin/streptavidin, biotin/avidin, protein A(G)/immunoglobulins, complementary segments of nucleic acids, virus/cellular receptor, lectin/carbohydrate, enzyme/inhibitor, enzyme/substrate. Also known are systems in which the binding partners form covalent bonds due to the presence of amino-reactive groups in such partners, such as succinimidyl esters, isothiocyanate, sulfonyl halides, or sulfhydryl reactive groups, including derivatives of haloacetates and maleimides, functional groups of click chemistry. Other binding partners may be halogens, steroids and 2,4-dinitrophenyls. For the purposes of the present description, the term "binding partner" refers to both the "recognition receptor" and its ligand. In some embodiments of the invention, there is a first and a second (corresponding to the first) binding partner, forming a molecular interface for binding (conjugation) of two objects to each other. That is, Covalent chemistry systems that allow two objects to bind to each other also fall under this term (see the systems described in Bioconjugate Techniques, Greg T. Hermanson. Academic Press, San Diego, CA. 1996 and its reprints).

4) The term "nucleic acid" means a polymer or oligomer consisting of nucleotides or compounds imitating them. The term also refers to oligonucleotides having a non-naturally occurring portion of the chain, but exhibiting the same properties as oligonucleotides of natural origin. It should be obvious to those skilled in the art that a wide variety of assays for detecting target nucleic acid sequences can be carried out in accordance with the present invention. The term "nucleotide" herein refers to ribonucleotides, deoxyribonucleotides, or nucleotide analogs having a chemical modification in one or more of the nucleotide building blocks (nitrogenous bases - purine or pyrimidine, sugar, phosphates). Modifications can be, for example, carried out at the fifth position of a pyrimidine, or at the eighth position of a purine, in the exocyclic amino group of cytosine, by replacing uracil with 5-bromouracil, replacing the hydroxy group at the 2'-position of a sugar with a halo group (either R, OR, SH, H, SR, NR ₂ , NH ₂ , NHR, CN). The term also applies to ribonucleic acids occurring in living organisms, either with a replaced base (xanthine, ionosine, etc.) or sugar (2'-methoxyribose, etc.). Modification of the phosphodiester bond is also possible, for example, chain formation with phosphorothionates, methylphosphonates and peptides. "Nucleic acid" can refer to a single- or double-stranded molecule consisting of monomers (nucleotides) that include fragments of sugars, phosphates, and purines or pyrimidines. In plants, animals, lower eukaryotes, and bacteria, "ribonucleic acid" (RNA) performs translation functions, i.e., serves as a transmitter of genetic information stored in "deoxyribonucleic acid" (DNA). The term "fragment" refers to a specific part of a DNA sequence.

5) The method of analysis and its hardware implementation related to the present invention can detect a target nucleic acid or other substances (collectively, analyte) contained in various samples. The term "sample" herein refers to any liquid potentially containing analytes. The sample can be obtained from human or other animal tissues or liquids (blood/blood serum, urine, saliva, tears, sputum, sweat, mucus, stool), from tissue cell culture samples, histological samples, biopsy samples, etc. The sample can also be obtained from agricultural products, bacteria, viruses, or their waste products and processing. Various garbage and waste, their processed products, drinking and waste water, processed or raw food products, air, etc. can also serve as a sample. If this invention is implemented for detecting desired nucleic acid sequences, it can be used to analyze genetic abnormalities and many other diseases, serve to monitor the progress of treatment of infectious diseases, and for many other applications.

6) The term “recognition oligonucleotide” refers to an NC containing a sequence complementary to the analyzed (target) nucleic acid (analyte).

7) The term "signal-generating substrate" refers to a substance that can interact with a signaling receptor and generate a detectable signal. For example, ABTS/TMB for peroxidase, BCIP/MBT for alkaline phosphatase, light for fluorescent compounds.

8) The term "signal receptor", "signal-producing reporter", "reporter" means a substance capable of detecting the analyte of interest through the release of energy or by enzymatic activity, such as fluorescent and chemiluminescent fragments, particles, enzymes, radioactive labels, light-emitting domains or molecules. For the purposes of this description, the mention of these examples means the possibility of replacing them with other similar substances-"signal receptors".

9) The term "porous material" or "chromatographic material" means a material having pores of at least 0.1 µm, preferably at least 10.0 µm, of sufficient size to allow liquid media to move through it by capillary forces.

10) The term "signal-producing system" denotes a set of reagents necessary for generating a detectable signal under the influence of external influence. As a rule, such systems generate a signal that can be detected by external means - for example, by measuring electromagnetic radiation, or by visual observation. Basically, such systems consist of a chromophore substrate and an enzyme that converts the substrate into a dye that absorbs electromagnetic radiation of a certain range, phosphorescent or fluorescent substances.

11) The location of the hairpin from the first or second end of the recognizing oligonucleotide at a distance of X nucleotide bases implies the following. The site of binding of the nanoparticle or signal receptor to the recognizing oligonucleotide should be considered the nucleotide with which their bond is formed (preferably covalent). In this case, if this nucleotide is the first in the hairpin (i.e. forms a non-covalent bond with another nucleotide), then the distance X = 0. If the hairpin begins with the adjacent nucleotide, then X = 1, etc.

12) The first/second end of a single-stranded recognition oligonucleotide refers not to the actual end of the molecule, but to one or the other of its sides. That is, the nucleotide base of the recognition oligonucleotide corresponding to the binding site of the signal receptor or nanoparticle is preferably located at the edge of the oligonucleotide, but may not be the outermost base in the oligonucleotide. The nucleotide base of the recognition oligonucleotide corresponding to the binding site of the signal receptor (or nanoparticle) refers to the monomer of the nucleic acid nucleotide (including nucleoside, saccharide, and phosphoric acid residue) to which the signal receptor (or nanoparticle) is bound.

13) In this description, the concepts of "receptor" and "ligand" are interchangeable and can be used to designate individual molecules in the above-described concept of "binding partners". For example, if in the literature in pairs streptavidin-biotin, antibody-antigen, the receptor is often understood to mean streptavidin and antibody, and the ligand is understood to mean biotin and antigen, then in the description of this invention the receptor can be understood to mean both streptavidin with antibody and biotin with antigen (in this case the ligand will be biotin with antigen and streptavidin with antibody, respectively).

14) A nanoparticle means a particle in a wide range - from 1 nm to 100 µm, preferably from 1 nm to 10 µm, preferably from 1 nm to 1 µm, preferably from 1 nm to 100 nm. In this case, nanoparticles can be metallic, polymeric, core-shell structures, etc.

15) The term "nanoparticles" means supramolecular structures that consist of more than one molecule. In this case, although nanoparticles are often used to denote objects up to 100 nm, within the framework of the description of this invention, objects larger than 100 nm are also meant - preferably up to 5 μm, but in some cases a size of up to 100 μm is acceptable. Thus, nanoparticles are understood to mean both nanoparticles and microparticles, microspheres, etc. In addition, particles with the mentioned dimensions are meant, both in all dimensions and in at least one.

16) Therapeutic means/molecules/agents. Therapeutic molecules/agents may be understood as substances, substances, molecules, supramolecular agents, nanoparticles, microparticles, nanoagents, microagents, formulations, dosage forms, etc., which are used both for therapy and diagnostics of various diseases or conditions of the body or patient, including labels, contrast agents, controlled-release components, paramagnetic and magnetic agents, cytotoxic agents, etc.

17) A "label" or "detectable element" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, commonly used labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin or haptens and proteins or other entities that can be detected, for example, by incorporating a radioactive label into a peptide or antibody specifically reactive with a target peptide. Any method known in the art for conjugating an antibody to a label can be used, for example, using the methods described in (Hermanson, G. T., Academic press, 2013).

18) As used herein, the term "pharmaceutical" refers to a composition that is useful in treating a disease or symptom of a disease.

19) The term "treating" or "therapy" refers to any evidence of success in treating or improving a disease, injury, pathology, or condition, including any objective or subjective parameters such as reduction; remission; lessening of symptoms or slowing of deterioration of a patient's condition, pathology, or condition; slowing the rate of degeneration; making the final stage of a disease less painful; improving the physical or mental well-being of a patient. Treatment or improvement of symptoms may be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric examinations, and/or psychiatric evaluation. For example, certain methods presented herein may successfully treat cancer by reducing the incidence of cancer and inducing remission of cancer. The term "treatment" and its extensions include prevention of an injury, pathology, condition, or disease.

20) “Disease” or “condition” refers to the health condition of a patient or subject that is treatable by the various drugs, dispersions, or other methods provided herein.

21) As used herein, the term "contrast agent"/"contrast medium" refers to a composition that, when administered to a subject, improves the detection limit or detection capability of a physical method, technique, or medical imaging device (e.g., radiographic instrument, X-ray, CT, PET, MRI, ultrasound, and other methods). The contrast agent can enhance the magnitude of signals associated with various structures or fluids within the subject.

22) "Patient" or "subject in need thereof" refers to a living organism suffering from or (possibly) susceptible to a disease or condition that can be treated by administration of a drug, pharmaceutical composition or agent as described herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goats, sheep, cows, deer and other non-mammalian animals. In particular, a patient may be a human.

23) As used herein, the term "controlled release component" or "externally activated agent" refers to a compound that, when combined with a composition as described herein (including embodiments), releases the composition at a controlled rate under conditions of use (in a patient). Such compounds include high molecular weight, anionic mucomimetic polymers, gel-forming polysaccharides, and finely divided drug carrier substrates, among others. These components are discussed in more detail in U.S. Patents 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are hereby incorporated by reference in their entirety for all purposes. In some embodiments, the "controlled release component" may be a delayed release, sustained action, extended release, timed release, synchronized release, controlled release, modified release, or a sustained release compound. In some embodiments, the compound disintegrates at the site of administration (e.g., subcutaneous, intravenous) or within the digestive tract (e.g., stomach, intestine) if the compound and composition are administered orally. In some embodiments, the controlled release component is a polymer and may be referred to as a "controlled release polymer." Additionally, an externally activated agent may be activated (e.g., release a therapeutic agent or bind to a target) by exposure to an external magnetic/electric field, ultrasound, light, electron beam, radioactive decay of any atoms, interaction with a chemical microenvironment, etc.

24) As used herein, the term "paramagnetic agent"/"paramagnetic means" refers to a paramagnetic compound useful in diagnostic imaging techniques (e.g., magnetic resonance imaging) as a contrast agent. In some embodiments, the paramagnetic agent comprises gadolinium, iron oxide, platinum iron, or manganese.

25) Cytotoxic agent (toxin) options include, but are not limited to, ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoside, vincristine, vinblastine, colchicine, dihydroxyanthracinatedione, actinomycin D, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme.

26) A non-exhaustive list of immunoassays includes: competitive and non-competitive formats, enzyme-linked immunosorbent assays (ELISA), microdot assays, Western blots, gel filtration and chromatography, immunochromatography, immunohistochemistry, flow cytometry or fluorescently labeled cell sorting (FACS), microarrays, etc. Such methods can also be used in situ, ex vivo, in vitro or in vivo, such as for diagnostic imaging.

It should be noted that throughout the application, alternatives are written in Markush groups, such that each position of the therapeutic agent may contain more than one possible therapeutic agent. In particular, it is intended that each member of the Markush group should be considered separately, thereby containing a different embodiment, and the Markush group should not be read as a single whole.

Detailed description of the invention

Embodiments of the present invention will now be described more fully with reference to the accompanying drawings, which show some, but not all, embodiments of the invention.

Within the framework of this invention, the following is proposed:

A method of optically controlled theranostics that includes the following steps:

i) select the luciferase enzyme and its substrate,

ii) select a therapeutic molecule,

iii) selecting nanoparticles capable of sorbing said substrate and said therapeutic molecule,

iv) mixing and incubating said nanoparticles with a solution of said substrate of said luciferase to sorb the substrate into the nanoparticle,

v) mixing and incubating said nanoparticles with a solution of said therapeutic molecule for its sorption into the nanoparticle,

vi) conjugating said nanoparticles with said luciferase enzyme,

vii) washing off said substrate that is not bound to said nanoparticle and obtaining theranostic autonomously bioluminescent nanoparticles,

viii) induce cytoblockade of the macrophage phagocytic system in the body by introducing antibodies against the erythrocytes of the said organism,

ix) introduce into the body the mentioned theranostic autonomously bioluminescent nanoparticles,

x) detecting a bioluminescent signal from said autonomously bioluminescent nanoparticles to optically monitor the localization of delivery of said nanoparticles carrying said therapeutic molecule in the body.

In addition, the method in which the mentioned nanoparticles are pre-functionalized with a first binding partner, the luciferase is functionalized with a second binding partner, and the process of conjugation of the nanoparticle with the luciferase is realized due to the interaction of the first and second binding partners and the formation of a bond between them.

In addition, the method in which the said steps are performed in the following sequence:

i) selecting the luciferase enzyme and its substrate, as well as nanoparticles capable of sorbing the said substrate,

ii) functionalizing said nanoparticles with a first binding partner and said luciferase with a second binding partner,

iii) mixing and incubating said functionalized nanoparticles with a solution of a substrate of said luciferase to sorb the substrate into the nanoparticle,

iv) conjugating said nanoparticles with the luciferase enzyme by conjugating said functionalized nanoparticles with said functionalized luciferase enzyme under binding conditions of said first and second binding partners, and the step of said washing of the substrate not bound to the nanoparticle is performed either after step (iii) or after step (iv).

In addition, a method in which said first and second binding partners are biotin and streptavidin.

In addition, a method in which said step of mixing and incubating said nanoparticles with a solution of said substrate is performed after said step of conjugating said nanoparticles with said luciferase enzyme.

In addition, a method in which said step of mixing and incubating said nanoparticles with a solution of said substrate is performed before said step of conjugating said nanoparticles with said luciferase enzyme.

In addition, a method in which luciferase-nanoparticle conjugates are washed from unbound luciferase.

In addition, a method is used in which nanoparticles are selected that are capable of sorbing the substrate of the mentioned luciferase with a substrate mass loading of more than 0.1% of the mass of the nanoparticle.

In addition, a method in which nanoparticles capable of sorbing the substrate of the mentioned luciferase are selected with a substrate mass loading of more than 1% of the nanoparticle mass.

In addition, a method in which nanoparticles capable of sorbing the substrate of the mentioned luciferase are selected with a substrate mass loading of more than 10% of the nanoparticle mass.

In addition, the method in which said nanoparticles are polymeric nanoparticles.

In addition, the method in which said nanoparticles are metal-organic framework polymer particles.

In addition, the method in which the said nanoparticles are nanoparticles based on a copolymer of lactic and glycolic acids.

Furthermore, a method in which said nanoparticles are magnetic nanoparticles coated with carboxymethyldextran.

In addition, the method in which the said luciferase is Nanoluc luciferase.

In addition, the method in which the said substrate is one of the following: furimazine, coelenterazine or their analogues.

In addition, a method in which the nanoparticle is additionally conjugated with a receptor for targeting said autonomously bioluminescent nanoparticles to a specific target.

Also, a method in which the said luciferase is a mixture of several luciferases.

Also a method in which said substrate is a mixture of several substrates.

Furthermore, a method in which said receptor or second receptor (recognition) is a lectin, antibody, or other "binding partner" of the analyte.

In addition, a method in which said cell sample is biopsy material.

In addition, a method in which said cell sample is a histological section.

Furthermore, a method wherein said cell sample is a suspended cell sample.

In addition, a method in which the said bioluminescent signal is detected (emitted) at emission wavelengths of 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 750-800, 800-850, 850-900, 900-950.

In addition, the method in which the emission is in the range of 400-525 nm.

In addition, the method in which the emission is in the range of 525-700 nm.

Furthermore, a method in which said molecular marker is a cellular marker.

In addition, the method, wherein said cellular marker is selected from the series: EGFR, HER2, PD-L1, EpCAM, CEA, PSA, CA-125, CA 19-9, CA 15-3, CA 72-4, MUC1, CD19, CD20, CD44, CD133, CD166, CD24, CD47, CD117, DR-5.

In addition, a method and/or nanolabel used in the diagnosis of diseases or conditions of the body.

Furthermore, a method in which said cellular marker is a molecular marker expressed in the cytoplasm of a cell.

In addition, a method and/or nanolabel used in gene therapy of diseases or conditions of the body.

In addition, a method in which said analyte is a small molecule.

In addition, a method wherein said analyte is a protein.

In addition, a method in which said analyte is a nucleic acid.

In addition, the method in which said test strip is an immunochromatographic test strip, a thin layer or an affinity chromatographic test strip.

In addition, the method in which the said test strip contains a nitrocellulose membrane.

In addition, a method in which the detection of the amount of particles of said carbon quantum dots retained on the test line is carried out within less than 2, 3, 5, 7, 10, 15, 20 minutes after the start of the analysis.

In addition, the method in which the said washing is carried out by ultrafiltration.

In addition, the method in which the said washing is carried out by centrifugation.

In addition, the method in which said luciferase is one of: nanoLuc luciferase, firefly luciferase, Renilla luciferase, Gaussia luciferase.

In addition, a method in which said luciferase is a mixture of several luciferases.

Furthermore, a method in which said substrate is a substrate selected from the group: a chromogenic substrate, a fluorogenic substrate, or a chemiluminescent substrate.

Furthermore, a method in which said antibody is selected from the group: aminoglobulin, immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), immunoglobulin G3 (IgG3), immunoglobulin G4 (IgG4), immunoglobulin M, immunoglobulin A, immunoglobulin E, immunoglobulin D, Fab antibody fragment, Fab' antibody fragment, Fab'2 antibody fragment, F(ab')2 antibody fragment, scFv antibody fragment, nanobody, scFv-Fc fragments, scFv-scFv (diabody), bispecific scFv fragments, Fab-scFv antibody fragment.

In addition, a method in which the said antibody is a genetically engineered analogue from the group: avimers, adgyrons, adnectins, monobodies, atrimers, anticalins, affibodies, affilins, affimers, affitins, nanophytins, darpins, knottins, obodis, Kunitz domain-based polypeptides, pronectins, repbodys, phynomers, centirins, ADAPT (ABD-Derived Affinity Proteins), NanoCLAMP (nano-CLostridial Antibody Mimetic Proteins), ARM (Armadillo repeat proteins), PDZ proteins.

In addition, a method for synthesizing autonomous bioluminescent nanoparticles, which includes the following steps:

i) select nanoparticles capable of sorbing small molecules,

ii) conjugate said nanoparticles with the enzyme luciferase,

iii) mixing and incubating said nanoparticles with a solution of a substrate of said luciferase to sorb the substrate into the nanoparticle,

iv) the substrate not bound to the nanoparticle is washed off and autonomously bioluminescent nanoparticles are obtained.

In addition, the method includes the following steps:

i) select nanoparticles capable of sorbing small molecules,

ii) conjugate said nanoparticles with the enzyme luciferase,

iii) mixing and incubating said nanoparticles or the resulting nanoparticle-luciferase conjugate in the case of step (iii) before step (iii) with a solution of the substrate of said luciferase for sorption of the substrate into the nanoparticle,

iv) the substrate not bound to the nanoparticle is washed off and autonomously bioluminescent nanoparticles are obtained.

In addition, the method includes the following steps:

i) polymer nanoparticles capable of sorbing small molecules are selected,

ii) conjugate said nanoparticles with the luciferase enzyme directly or through an additional biomolecular interface,

iii) mixing and incubating the resulting conjugates with a solution of the substrate of the said luciferase to adsorb the substrate into the conjugate nanoparticles,

iv) the substrate not bound to the conjugates is washed away and autonomously long-term bioluminescent nanoparticles are obtained,

characterized in that they do not require the addition of a substrate when using nanoparticles.

In addition, the method includes the following steps:

i) select nanoparticles capable of sorbing small molecules,

ii) conjugate said nanoparticles with the enzyme luciferase,

iii) mixing and incubating the resulting conjugates with a solution of the substrate of the said luciferase to adsorb the substrate into the conjugate nanoparticle,

iv) the substrate not bound to the conjugate is washed away and autonomously bioluminescent nanoparticles are obtained.

In addition, the method includes the following steps:

i) select nanoparticles capable of sorbing small molecules,

iii) mixing and incubating said nanoparticles with a solution of a substrate of said luciferase to sorb the substrate into the nanoparticle,

iv) the substrate that has not bound to the nanoparticle is washed away,

ii) conjugate said nanoparticles with the enzyme luciferase,

optionally iv) washing off the luciferase that is not bound to the nanoparticle,

and obtain autonomously bioluminescent nanoparticles.

In addition, a method in which there are additional washing steps between the specified steps.

In addition, a method in which conjugates of nanoparticles with luciferase are additionally washed from unbound luciferase after conjugation of said nanoparticle with the luciferase enzyme.

In addition, a method in which said luciferase is Firefly luciferase.

In addition, a method wherein said substrate is luciferin.

Furthermore, a method in which said substrate is a mixture of luciferin and ATP.

In addition, a method in which said substrate is a mixture of luciferin, ATP, coenzyme A.

In addition, a method in which the obtained autonomous bioluminescent nanoparticles produce detectable light for more than 10 minutes, more preferably 30 minutes, more preferably 1 hour, more preferably 3 hours, more preferably 10 hours, more preferably 24 hours, more preferably 48 hours, more preferably 72 hours.

In addition, a method in which the substrate not bound to the nanoparticle is washed so that the concentration of the substrate in the supernatant is reduced by more than 10 times, preferably 30 times, preferably 100 times, preferably 300 times, preferably 1000 times, preferably 10000 times, preferably 100000 times, preferably 1000000 times, preferably 1,000,000 times compared to the concentration of the substrate in the solution during said mixing of said nanoparticles with the substrate solution of said luciferase.

In addition, a method in which nanoparticles are selected that are capable of sorbing the substrate of the said luciferase with a substrate weight loading of more than 0.001%, preferably more than 0.01%, preferably more than 0.1%, preferably more than 1%, preferably more than 3%, preferably more than 10%, preferably more than 20%, preferably more than 30%, preferably more than 40%, preferably more than 50% of the nanoparticle weight.

In addition, a method in which the said incubation is carried out for at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 120 minutes, at least 180 minutes, at least 480 minutes.

In addition, a method in which the said incubation is carried out for no more than 1 minute, no more than 2 minutes, no more than 5 minutes, no more than 10 minutes, no more than 20 minutes, no more than 30 minutes, no more than 45 minutes, no more than 60 minutes, no more than 120 minutes, no more than 180 minutes, no more than 480 minutes.

Furthermore, a method in which, in the step of conjugation with luciferase, the nanoparticle is further conjugated with a receptor for targeting said autonomously bioluminescent nanoparticles to a specific target.

In addition, a method in which nanoparticles are selected that are capable of sorbing the said substrate reversibly.

In addition, a method in which said luciferase is nanoLuc luciferase, firefly luciferase, thermostable luciferase, Renilla luciferase, Gaussia luciferase or genetically engineered mutated forms thereof.

In addition, a method for detecting an analyte in a sample, including the following steps:

i) select nanoparticles capable of sorbing small molecules,

ii) conjugate said nanoparticles with the enzyme luciferase,

iii) conjugating said nanoparticles with a receptor for the analyte or a molecular analogue of the analyte,

iv) mixing and incubating said nanoparticles with a solution of a substrate of said luciferase to sorb the substrate into the nanoparticle,

v) the substrate not bound to the nanoparticle is washed off and functionalized autonomously bioluminescent nanoparticles are obtained,

vi) mixing said sample with a suspension of said functionalized autonomously bioluminescent nanoparticles,

vii) applying the resulting mixture to a test strip, in the “test line” zone of which a specific receptor is immobilized, capable of recognizing and/or binding to the analyte or competing with the analyte for binding to the said receptor to the analyte of the said functionalized autonomously bioluminescent nanoparticles,

viii) assessing the content of said analyte in the sample by recording the bioluminescent signal from said functionalized autonomously bioluminescent nanoparticles retained on the test line.

In addition, a method in which said analyte is a small molecule.

In addition, a method wherein said analyte is a protein.

In addition, a method in which said analyte is a nucleic acid.

In addition, the method in which said test strip is an immunochromatographic test strip, a thin layer or an affinity chromatographic test strip.

In addition, the method in which the said test strip contains a nitrocellulose membrane.

In addition, a competitive solid-phase assay method for detecting an analyte in a sample, comprising the following steps:

i) select nanoparticles capable of sorbing small molecules,

ii) conjugate said nanoparticles with the enzyme luciferase,

iii) conjugate said nanoparticles with a receptor for the analyte,

iv) mixing and incubating said nanoparticles with a solution of a substrate of said luciferase to sorb the substrate into the nanoparticle,

v) the substrate not bound to the nanoparticle is washed off and functionalized autonomously bioluminescent nanoparticles are obtained,

v) mixing said sample with said functionalized autonomously bioluminescent nanoparticles,

ii) the resulting mixture is brought into contact with the surface of a solid phase carrying molecules of the analyte or its molecular analogue,

iii) removing said autonomously bioluminescent nanoparticles that are not bound to said solid phase surface,

v) recording the bioluminescent signal from the solid phase-bound said autonomously bioluminescent nanoparticles as a measure of the analyte content in the sample.

In addition, a method in which the surface of the solid phase is the surface of a multi-well plate.

Furthermore, a method in which the surface of the solid phase is the surface of a 96-, 384-, or 1536-well plate.

Furthermore, a method in which said receptor is selected from the group: a monoclonal antibody, a polyclonal antibody,

Furthermore, a method in which said receptor is selected from the group: amimmunoglobulin, immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), immunoglobulin G3 (IgG3), immunoglobulin G4 (IgG4), immunoglobulin M, immunoglobulin A, immunoglobulin E, immunoglobulin D, Fab antibody fragment, Fab' antibody fragment, Fab'2 antibody fragment, F(ab')2 antibody fragment, scFv antibody fragment, nanobody, scFv-Fc fragments, scFv-scFv (diabody), bispecific scFv fragments, Fab-scFv antibody fragment.

Furthermore, a method in which said receptor is selected from the group: avimers, adgyrons, adnectins, monobodies, atrimers, anticalins, affibodies, affilins, affimers, affitins, nanophytins, darpins, knottins, obodis, Kunitz domain-based polypeptides, pronectins, repbodys, phynomers, centirins, ADAPT (ABD-Derived Affinity Proteins), NanoCLAMP (nano-CLostridial Antibody Mimetic Proteins), ARM (Armadillo repeat proteins), PDZ proteins.

Furthermore, a method in which said molecular analogue of the analyte is a carrier protein conjugated to the analyte molecules.

In addition, a solid-phase analysis method for detecting an analyte in a sample, comprising the following steps:

i) contacting said sample with the surface of a solid phase bearing a receptor for the analyte,

ii) select nanoparticles capable of sorbing small molecules,

iii) conjugate said nanoparticles with the enzyme luciferase,

iv) conjugating said nanoparticles with a receptor for the analyte,

v) mixing and incubating said nanoparticles with a solution of the substrate of said luciferase to sorb the substrate into the nanoparticle,

v) the substrate not bound to the nanoparticle is washed off and functionalized autonomously bioluminescent nanoparticles are obtained,

v) said functionalized autonomously bioluminescent nanoparticles are contacted with said solid phase surface,

iii) removing said functionalized autonomously bioluminescent nanoparticles that are not bound to said solid phase surface,

v) recording the bioluminescent signal from the solid phase-bound said autonomously bioluminescent nanoparticles as a measure of the analyte content in the sample.

In addition, a bioimaging method that includes the following steps:

i) select nanoparticles capable of sorbing small molecules,

ii) conjugate said nanoparticles with the enzyme luciferase,

iii) mixing and incubating said nanoparticles with a solution of a substrate of said luciferase to sorb the substrate into the nanoparticle,

iv) the substrate not bound to the nanoparticle is washed off and autonomously bioluminescent nanoparticles are obtained,

v) introduce the said autonomously bioluminescent nanoparticles into the body,

vi) detecting a bioluminescent signal from said autonomously bioluminescent nanoparticles.

Also, a method used in diagnosing diseases or conditions of the body.

In addition, a method of microscopy of a cell sample, which includes the following steps:

i) select nanoparticles capable of sorbing small molecules,

ii) conjugate said nanoparticles with the enzyme luciferase,

iii) mixing and incubating said nanoparticles with a solution of a substrate of said luciferase to sorb the substrate into the nanoparticle,

iv) the substrate not bound to the nanoparticle is washed off and autonomously bioluminescent nanoparticles are obtained,

v) incubating the obtained said autonomously bioluminescent nanoparticles with the cellular sample,

vi) the obtained samples are analyzed using optical microscopy by detecting the bioluminescent signal of the said autonomously bioluminescent nanoparticles.

Furthermore, a method in which said nanoparticles are further conjugated with a receptor against a molecular marker in a cellular sample.

In addition, a method in which the obtained samples are analyzed using optical microscopy by detecting a bioluminescent signal of said autonomously bioluminescent nanoparticles as a measure of the presence of a molecular marker in said cellular sample.

In addition, a method in which the obtained samples are analyzed using optical microscopy by detecting a bioluminescent signal of said autonomously bioluminescent nanoparticles for localizing a molecular marker in said cellular sample.

Furthermore, a method in which said nanoparticles are further conjugated with a receptor against a molecular marker in a cellular sample.

In addition, the method, wherein said cellular marker is selected from the series: EGFR, HER2, PD-L1, EpCAM, CEA, PSA, CA-125, CA 19-9, CA 15-3, CA 72-4, MUC1, CD19, CD20, CD44, CD133, CD166, CD24, CD47, CD117, DR-5.

In addition, a method in which the obtained samples are analyzed using optical microscopy by detecting a bioluminescent signal of said autonomously bioluminescent nanoparticles as a measure of the presence of a molecular marker in said cellular sample.

In addition, a method in which the obtained samples are analyzed using optical microscopy by detecting a bioluminescent signal of said autonomously bioluminescent nanoparticles for localizing a molecular marker in said cellular sample.

In addition, a method in which said cell sample is biopsy material.

In addition, a method in which said cell sample is a histological section.

Furthermore, a method wherein said cell sample is a suspended cell sample.

Furthermore, a method in which said molecular marker is a cellular marker.

Furthermore, the method, wherein said luciferase is selected as NanoLuc luciferase, and the fluorescent protein is selected from the series: aacuGFP1, aceGFP, afraGFP, amFP506, anm1GFP1, anobCFP1, anobCFP2, AvicFP1, AzamiGreen, bfloGFPa1, BrUSLEE, bsDronpa, cerFP505, cfSGFP2, CGFP, cgreGFP, CRISPRed2s, CyOFP1, deGFP2, Dronpa-2, Dronpa-3, dVFP, ECGFP, eCGP123, eechGFP1, efasCFP, efasGFP, EGFP, Emerald, Folding Reporter GFP, frSkylan-S, G1, G2, G3, GCaMP6f, GdT, gfasGFP, GFP (S65T), GFPmut2, GFPxm16, GFPxm18, GFPxm19, GGvT, GZnP3, htFuncLib_acid.1, htFuncLib_acid.2, htFuncLib_acid.3, htFuncLib_acid.4, htFuncLib_acid.5, htFuncLib_acid.6, htFuncLib_basic.2, htFuncLib_basic.3, htFuncLib_basic.4, htFuncLib_basic.6, htFuncLib_bleach.1, htFuncLib_bleach.2, htFuncLib_bleach.4, htFuncLib_bleach.5, htFuncLib_bleach.6, htFuncLib_em-blue-shift.1, htFuncLib_em-blue-shift.2, htFuncLib_fast.1, htFuncLib_fast.2, htFuncLib_fast.3, htFuncLib_fast.4, htFuncLib_mid.10, htFuncLib_mid.11, htFuncLib_mid.12, htFuncLib_mid.13, htFuncLib_mid.2, htFuncLib_mid.4, htFuncLib_mid.7, htFuncLib_mid.8, htFuncLib_mid.9, htFuncLib_sf:acid.3, htFuncLib_sf:acid.4, htFuncLib_sf:acid.5, htFuncLib_sf:bleach.1, htFuncLib_sf:bleach.2, htFuncLib_sf:bleach.3, htFuncLib_sf:em-blue-shift.1, htFuncLib_sf:em-blue-shift.2, htFuncLib_sf:fast.3, htFuncLib_sf:mid.10, htFuncLib_sf:mid.11, htFuncLib_sf:mid.12, htFuncLib_sf:mid.5, htFuncLib_sf:mid.7, htFuncLib_sf:mid.9, htFuncLib_sf:photostable.1, htFuncLib_sf:photostable.2, htFuncLib_slow.2, htFuncLib_slow.3, htFuncLib_slow.5, iq-mEmerald, IrisFP-M159A, KCY-R1, LanFP1, Lime, LSSmApple, LSS-mKate2, LSSmScarlet, LSSmScarlet2, LSSmScarlet3, LucY, mAvicFP1, mAzamiGreen, mBaoJin, mBaoJin-T55S, mBaoJin-V152E, mChartreuse, mCRISPRed, meffCFP, meffGFP, mEGFP, mEmerald, mEosFP-M159A, mGFPmut3, mHoneydew, MiCy, mMiCy, moxGFP, msGFP2, mStayGold, mStayGold2, mTFP1, mTurquoise2-1710, muGFP, mUkG, mVFP, mVFP1, mWasabi, NowGFP, oxGFP, oxStayGold, pdae1GFP, pmeaGFP1, pmeaGFP2, pmimGFP1, pmimGFP2, ppluGFP1, ppluGFP2, pporGFP, PROSS-eGFP, rsFolder, rsFolder2, sarcGFP, sg25, SGFP1, SGFP2, SGFP2(206A), SGFP2(E222Q), Skylan-NS, Skylan-S, StayGold, StayGold-E138D, stylGFP, superecliptic pHluorin, Superfolder GFP, TagGFP, TagGFP2, td5oxStayGold, td5StayGold, td8ox2StayGold, td8oxStayGold, tdStayGold, Thermostable Green Protein, TurboGFP, UnaG, usGFP, VFP, vsfGFP-0, vsfGFP-9, WasCFP, ZsGreen or their genetically engineered variants.

Furthermore, the method wherein said luciferase is selected from Firefly luciferase and the fluorescent protein is selected from the series: amilFP593, ccalRFP1, cgfTagRFP, dimer2, d-RFP618, DsRed, DsRed.M1, DsRed.T3, DsRed.T4, DsRed2, DsRed-Express, DsRed-Express2, DsRed-Max, dTomato, E2-Orange, E2-Red/Green, eqFP578, eqFP611, eqFP611 V124T, FR-1, frFAST, FusionRed-MQV, iq-mApple, KO, mApple, mBanana, mCherry-XL, meffRFP, mKO, mKOκ, mKO2, mLychee, mNectarine, mOrange, mOrange2, mPapaya, mRFP1-Q66C, mRFP1-Q66S, mRFP1-Q66T, mRuby, mRuby2, mRuby3, mScarlet, mScarlet-220A, mScarlet-2A, mScarlet-2A-84W, mScarlet3, mScarlet3-H, mScarlet-H, mScarlet-I, mScarlet-I-220A, mScarlet-I3, mScarlet-I3-NCwt, mTangerine, OFP, PAmCherry1, PAmCherry3, PATagRFP, PATagRFP1297, PATagRFP1314, pHuji, RFP611, RFP618, RRvT, scubRFP, super-TagRFP, TagRFP, TagRFP-T, tdimer2(12), td-RFP611, tdTomato, TurboRFP, or their genetically engineered variants.

2) The method according to item 1, wherein said therapeutic molecule is a nucleic acid from the series: DNA, RNA, plasmid DNA, matrix RNA, antisense DNA, antisense RNA, small interfering RNA, piwiRNA, microRNA, aptamer.

Furthermore, a method in which said therapeutic molecule is selected from the series: antibiotics, antiviral drugs, analgesics, anti-inflammatory agents, chemotherapeutic agents, enzyme inhibitors, tyrosine kinase inhibitors, hormones, synthetic hormonal analogues, peptides, proteins, therapeutic antibodies, siRNA, antisense oligonucleotides, mRNA, plasmids, recombinant proteins, cytokines, interferons, immunotherapeutic agents, cell therapies, chemical proteases, receptor blockers, radiopharmaceuticals.

Also, a method that is used in gene therapy for diseases or conditions of the body.

Also, a method that is used to diagnose diseases or conditions of the body.

In addition, the method, wherein said cellular marker is selected from the series: EGFR, HER2, PD-L1, EpCAM, CEA, PSA, CA-125, CA 19-9, CA 15-3, CA 72-4, MUC1, CD19, CD20, CD44, CD133, CD166, CD24, CD47, CD117, DR-5.

Furthermore, a method in which said cellular marker is a molecular marker expressed in the cytoplasm of a cell.

In addition, a method in which said sample is biopsy material.

In addition, a method in which said sample is a histological section.

Furthermore, a method wherein said sample is a suspended cell sample.

Furthermore, a method in which said nanoparticles are further conjugated with a receptor against a molecular marker in said sample.

Furthermore, a method in which said molecular marker is a cellular marker.

In addition, the method, wherein said cellular marker is selected from the series: EGFR, HER2, PD-L1, EpCAM, CEA, PSA, CA-125, CA 19-9, CA 15-3, CA 72-4, MUC1, CD19, CD20, CD44, CD133, CD166, CD24, CD47, CD117, DR-5.

In addition, a method in which the obtained samples are analyzed using optical microscopy by detecting the bioluminescent signal of said autonomously bioluminescent nanoparticles as a measure of the presence of a molecular marker in said sample.

In addition, a method in which the obtained samples are analyzed using optical microscopy by detecting a bioluminescent signal of said autonomously bioluminescent nanoparticles for localizing a molecular marker in said sample.

In addition, a method in which the said nanoparticles comprise or consist of a magnetic, fluorescent, protein (including one that is a cross-linked, polymerized, or aggregated protein), polymer, including one consisting of at least one of the following polymers: polystyrene, dextran, polypeptide, polylactide glycolic acid, or block copolymers) or crystalline (gold, silver, semiconductor, or metal) nano- or microparticle.

In addition, the method in which said nanoparticles are microparticles.

Furthermore, the method, wherein said analyte receptor is selected from the group: gamma immunoglobulin, immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), immunoglobulin G3 (IgG3), immunoglobulin G4 (IgG4), immunoglobulin M, immunoglobulin A, immunoglobulin E, immunoglobulin D, Fab antibody fragment, Fab' antibody fragment, Fab'2 antibody fragment, F(ab')2 antibody fragment, scFv antibody fragment, nanobodies, scFv-Fc fragments, scFv-scFv (diabodies), bispecific scFv fragments, Fab-scFv antibody fragment, avimers, adgyrons, adnectins, monobodies, atrimetres, anticalins, affibodies, affilins, affimers, affitins, nanophytins, darpins, knottins, obodis, Kunitz domain-based polypeptides, pronectins, repbodys, phynomers, centirins, ADAPT (ABD-Derived Affinity Proteins), NanoCLAMP (nano-CLostridial Antibody Mimetic Proteins), ARM (Armadillo repeat proteins), PDZ proteins.

Furthermore, a method in which said luciferase is selected from the series: nanoLuc luciferase, firefly luciferase, thermostable luciferase, Renilla luciferase, Gaussia luciferase.

Furthermore, the method in which the said nanoparticle is conjugated with a receptor against a molecular target selected from the group: amimmunoglobulin, immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), immunoglobulin G3 (IgG3), immunoglobulin G4 (IgG4), immunoglobulin M, immunoglobulin A, immunoglobulin E, immunoglobulin D, Fab antibody fragment, Fab' antibody fragment, Fab'2 antibody fragment, F(ab')2 antibody fragment, scFv antibody fragment, nanobodies, scFv-Fc fragments, scFv-scFv (diabodies), bispecific scFv fragments, Fab-scFv antibody fragment, avimers, adgyrons, adnectins, monobodies, atrimeters, anticalins, affibodies, affililines, affimers, affitins, nanophytins, darpins, knottins, obodis, Kunitz domain-based polypeptides, pronectins, repbodys, phynomers, centirins, ADAPT (ABD-Derived Affinity Proteins), NanoCLAMP (nano-CLostridial Antibody Mimetic Proteins), ARM (Armadillo repeat proteins), PDZ proteins.

In addition, a method in which said first and second binding partners are a pair of biotin and streptavidin.

In addition, a method in which said nanoparticles are pre-functionalized with a first binding partner, luciferase is functionalized with a second binding partner, and the process of conjugation of the nanoparticle with luciferase is realized by interaction of the first and second binding partners and formation of a bond between them, wherein said first and second binding partners are a pair of biotin and streptavidin.

Within the framework of this invention, autonomous luminescent nanoparticles were developed and their application for various biomedical applications was demonstrated. The invention is based on the phenomenon of bioluminescence, which consists of the emission of light in the visible range, arising from the interaction of luciferase enzymes and their substrates (luciferins). Autonomous bioluminescent nanoparticles were obtained by sorption of the substrate into nanoparticles and conjugation of the nanoparticles with luciferase. When the substrate was released from the particles, it began to react with luciferase, causing prolonged autonomous luminescence. Various combinations of luciferases and luciferins were tested: NanoLuc-furimazine, Renilla-coelenterazine and Gaussia-coelenterazine, etc. Among the studied enzymes, NanoLuc luciferase in combination with the furimazine substrate turned out to be the most effective. This enzyme exhibits high activity, resistance to external conditions and provides intense glow.

Various types of nanoparticles capable of efficiently sorbing luciferins were used as a platform for creating the luminescent system, including metal-organic coordination polymers (MOCPs), zeolites, silicon and carbon nanoparticles, polystyrene nanoparticles, nanoparticles made of the copolymer of lactic and glycolic acids (PLGA), chitosan and other polymers. The sorption capacities of different types of nanoparticles with respect to various luciferins were studied. Autonomous bioluminescent systems were successfully obtained based on a wide range of nanoparticles, including liposomes and lipid nanoparticles, where the encapsulation of the substrate occurred during synthesis, which reflects the versatility of this approach. However, the most effective sorbents were MOCPs, capable of sorbing amounts of substrate up to 50% of their own weight. Among a wide range of metal-organic frameworks, preference was given to multifunctional hybrid particles with a magnetite (Fe3O4) core and a shell made of MOFs of the MIL-100(Fe) structure. These particles also demonstrated high sorption capacity (up to 20%), as well as high biocompatibility and low toxicity. In addition to MOFs, biomedical applications of the invention were demonstrated using PLGA-based particles, as well as Magsphere polystyrene magnetic nanoparticles (PMNs) (including carboxylated ones). Scanning electron microscopy images of these nanoparticles are shown in Fig. 1.

Various approaches have been implemented to create luminescent nanoparticles. The main approach was to conjugate the nanoparticles with luciferase and then adsorb the substrate onto the nanoparticles. The modification of the nanoparticle surface by luciferase was confirmed by various physicochemical methods, including dynamic light scattering and spectroscopic methods. When conjugating the nanoparticles with luciferase, an increase in the hydrodynamic dimensions of the nanoparticles was observed, as well as the appearance of additional peaks in the infrared spectrum. The resulting luminescent nanoparticles had the ability to emit light for a long time under various physiological conditions (Fig. 2), including flow systems, cell samples, and in vivo models, which is due to the smooth nature of the substrate desorption. Alternative approaches have involved pre-sorption of the substrate and subsequent conjugation of the nanoparticles with luciferase using click chemistry or through additional linkers, such as interaction of streptavidin-coated nanoparticles with biotinylated enzyme. Such approaches have been used when additional low-molecular-weight therapeutic compounds need to be sorbed.

Next, the use of autonomous bioluminescent nanoparticles as a laboratory diagnostic tool for analyzing the content of low- and high-molecular compounds in a sample by immunochromatographic and solid-phase (including enzyme-linked immunosorbent assay) methods was demonstrated. The possibility of qualitatively determining the biotin content in a sample by competitive immunochromatographic assay in the express test format was demonstrated. In the absence of the analyte in the sample, a luminescent signal was observed in the test zone on the strip (Fig. 3-a), whereas in the presence of the analyte in the sample, no accumulation of nanoparticles in the test zone occurred (Fig. 3-b). Also, using bioluminescent nanoparticles, the possibility of quantitatively determining the concentration of chloramphenicol in a sample by competitive ELISA (Fig. 4) and troponin by direct ELISA with detection thresholds of 500 and 0.13 ng/ml, respectively, was demonstrated.

The proposed approach demonstrated the possibility of studying the interaction of nanoparticles with cells using microscopy and flow cytometry. Microscopic photographs of cells interacting with autonomous bioluminescent magnetic nanoparticles under the influence of an external magnetic field were obtained (Fig. 5). In places where nanoparticles accumulated, a luminescent signal was detected in the absence of external illumination, which can be used to modulate the magnetic delivery of small molecules or magnetofection. To improve the interaction of bioluminescent nanoparticles with cells, their surface was modified with a positively charged polymer - branched polyethyleneimine. The interaction of the obtained nanoparticles with cells was confirmed by flow cytometry by the shift of the fluorescent peak to an area with greater intensity compared to the control samples (Fig. 6).

The possibility of modifying the surface of nanoparticles with target molecules was also demonstrated, which made it possible to observe the specific interaction of bioluminescent nanoparticles with various molecular targets. The surface of nanoparticles was modified with antibodies to HER2 receptors, anti-HER2 affibody, DARPin G3, as well as various antibody fragments, including anti-CD4 Fab fragment, anti-CD3 F(ab')2 fragment, anti-CD8 scFv fragment. The possibility of specific interaction of modified nanoparticles with the corresponding target cells was demonstrated. Fig. 7 shows the results of the specificity test on HER2 positive and negative cell lines using bioluminescent PLGA-NanoLuc-trastuzumab nanoparticles. On EMT-HER2 cells, a more than twofold increase in the luminescent signal was observed with PLGA-NanoLuc-trastuzumab particles compared to PLGA-NanoLuc particles. In this case, no statistically significant difference in particle luminescence was observed on HER2-negative EMT6/P cells.

The proposed approach demonstrated the possibility of quantitatively studying the biodistribution of nanoparticles in the body using ex vivo analysis, as well as qualitatively analyzing the pharmacokinetics of nanoparticles in vivo. Bioluminescent nanoparticles based on PLGA, KPMNPs, and Fe3O4@MIL-100(Fe) were administered to mice intravenously. An hour later, a luminescent signal was observed only in the organs, indicating that the circulation of nanoparticles in the bloodstream had ceased. Analysis of the distribution of the luminescent signal by organs ex vivo allowed us to determine the percentage of bioluminescent nanoparticles in different parts of the body. The analysis showed that more than 60% of KPMNP-NanoLuc accumulated in the spleen (Fig. 8-a), Fe3O4@MIL-100(Fe)-KMD-NanoLuc predominantly entered the lungs and spleen (Fig. 8-b), and PLGA-NanoLuc - in the spleen, liver and lungs (Fig. 8-c). The data on the biodistribution of magnetic nanoparticles obtained by magnetometry (MPQ) were in good agreement with the luminescent distribution. At the same time, the analysis of the biodistribution of nanoparticles using fluorescent labels or using non-autonomous bioluminescent systems requiring a separate injection of the substrate showed lower sensitivity and accuracy compared to the control method.

The possibility of studying the kinetics of bioluminescent nanoparticle accumulation in organs was also demonstrated using Fe3O4@MIL-100(Fe)@KMD-NanoLuc as an example. After 15 seconds of circulation, a signal was detected in the lungs, and after 1 min - in the spleen (Fig. 9). The use of an autonomous luminescent system made it possible to detect the circulation of nanoparticles and accumulation in organs in real time, as well as with a high signal-to-noise ratio. Using bioluminescent nanoparticles conjugated with trastuzumab, the possibility of using nanoparticles for diagnostic purposes was demonstrated. Targeted nanoparticles gave a luminescent signal in a HER2-positive tumor, and accumulated significantly less effectively in HER2-negative tumors. Using this diagnostic approach, it is possible to detect the presence of oncological diseases and determine characteristic markers, which allows for the selection of more effective therapy.

In order to improve the biovisualization properties of nanoparticles, an approach was developed to shift the emission spectrum to the long-wavelength region. Living tissues absorb electromagnetic radiation in this range worse, which allows increasing the sensitivity of the proposed method. For this purpose, nanoparticles were additionally conjugated with fluorescent proteins whose excitation spectrum intersects with the spectrum of bioluminescent radiation. Luminescent nanoparticles containing NanoLuc luciferase were conjugated with various fluorescent proteins, including CyOFP1, TurboGFP and LSSmScarlet, excited in the wavelength range of 470-500 nm. And nanoparticles containing Firefly luciferase were conjugated with fluorescent proteins mScarlet, RFP611 and RFP618, excited in the wavelength range of 559-569 nm. The resulting autonomous bioluminescent BRET systems exhibited a shift in the emission spectrum to a longer wavelength region, which made it possible to detect smaller amounts of nanoparticles introduced into the mouse body, as well as to increase the detection sensitivity in in vitro applications where samples or other objects that absorb in the luciferin emission range are used.

In addition to the use of nanoparticles for bioimaging and diagnostics, the therapeutic potential of bioluminescent nanoparticles has also been demonstrated, which allows these nanoparticles to be called theranostic agents. It has been shown that in addition to the substrate, MOPC nanoparticles can efficiently sorb various other low-molecular therapeutic substances, including antitumor, antiviral, anti-inflammatory and antibacterial drugs, and also bind to high-molecular therapeutic nucleic acids and protein molecules. The ability of bioluminescent particles to deliver the chemotherapeutic agent doxorubicin to a tumor and simultaneously visualize the location of the tumor in the body has been demonstrated. To improve the therapeutic potential of nanoparticles, the technology of cytoblockade of the mononuclear phagocyte system was used, which consists of preliminary introduction of anti-erythrocyte antibodies into the body. This approach allows to temporarily saturate the immune system, thereby increasing the time of circulation of nanoparticles in the bloodstream, which contributes to more effective accumulation of nanoparticles in target organs. Due to the use of the proposed technology, a more intense luminescent signal in the tumor was observed, as well as a more significant therapeutic effect of nanoparticles. In addition, a magnet was applied to the tumor grafting site, which significantly increased the accumulation of magnetic nanoparticles in the tumor, as well as statistically significantly extended the life of mice compared to non-magnetic particles. A decrease in the toxicity of therapy was also observed compared to free doxorubicin according to biochemical indicators.

It should be noted that there is a great advantage in using multimodal and multifunctional nanoagents created – magnetic, radiating, and/or loaded with therapeutic molecules – in theranostics and other applications described. Due to additional properties, the accuracy of particle localization, treatment synergy, etc. are significantly improved.

Various aspects

The detailed descriptions described above do not limit the range of possibilities and applications of the invention, but are only given for convenience.

Types of nanoparticles capable of sorbing small molecules (including luciferase substrates)

Nanoparticles capable of sorbing small molecules are used in various fields, including water purification, biomedicine and catalytic processes. Their efficiency is determined by particle size, surface structure and chemical properties. Sorption efficiency depends on the nature of the molecule (hydrophilic/hydrophobic, charge, size) and the properties of the nanoparticle:

1. Carbon nanomaterials

• Nanotubes : Have a large surface area and high adsorption capacity. Used for the sorption of organic and inorganic molecules.

• Graphene and its oxides : Due to their two-dimensional structure and functional groups, they can effectively adsorb molecules such as metals, organic pollutants and gases.

2. Metal oxide nanoparticles

• Zinc Oxide (ZnO) and Titanium Oxide (TiO₂) : Are widely used to remove organic molecules and pollutants from water due to their photocatalytic properties.

• Iron oxide (Fe₂O₃, Fe₃O₄) : Can absorb heavy metals and organic substances; have magnetic properties.

3. Metallic nanoparticles

• Gold (Au) and silver (Ag) : Due to the high reactivity of the surface, they are able to absorb molecules such as sulfur, oxygen or organic compounds.

• Copper (Cu) nanoparticles : Used to remove toxic molecules such as ammonia or heavy metal ions.

4. Zeolites and porous materials

• Nanoporous zeolites (aluminosilicates) have an ordered pore structure that is ideal for the sorption of small molecules such as gases and organic substances.

5. Metal-organic frameworks (MOFs) or metal-organic framework polymers (MOFs)

• MOFs are a combination of metals and organic ligands that provide high surface area and controlled porosity. They are particularly effective for the sorption of gases (CO₂, CH₄) and volatile organic compounds.

6. Nanoparticles with functionalized surface

• Modification with chemical groups (e.g. amino groups, thiols, carboxyl groups) increases selectivity for certain molecules.

7. Polymer nanoparticles:

A. Nanogels

Description: Three-dimensional cross-linked polymer networks that can swell in aqueous environments.

Example: Poly(acrylic acid), poly(N-isopropylacrylamide) (PNIPAM).

Application: Drug delivery, toxin sorption, catalysis.

B. Nanocapsules

Description: Hollow particles with a polymer shell and a core that may contain an active substance.

Example: Polycaprolactone (PCL), Polylactide-co-glycolide (PLGA).

Application: Controlled release of drugs, transport of hydrophobic molecules.

V. Nanospheres

Description: Solid polymer particles without cavities that evenly distribute active substances throughout their structure.

Example: Polystyrene, poly(methyl methacrylate) (PMMA).

Application: Biosensors, sorbents, catalytic materials.

G. Block copolymer nanoparticles

Description: Particles obtained from block copolymers with different chemical blocks. Can form micelles or other nanostructures.

Example: Polystyrene-b-polyethylene glycol (PS-PEG).

Application: Delivery of hydrophobic/hydrophilic molecules, stabilization of emulsions.

D. Dendrimers

Description: Branched polymer structures with a high density of functional groups on the surface.

Example: Polyamidoamine (PAMAM) dendrimers.

Application: Targeted drug delivery, metal sorption.

E. Molecularly imprinted polymers (MIPs)

Description: Polymers synthesized using a template molecule that creates "fingerprints" for selective binding of specific molecules.

Example: Polymethacrylate crosslinked with acrylic monomers.

Application: Selective sorption of toxins, hormones, drugs.

G. Stimulus-responsive nanoparticles

Description: Particles that react to changes in the environment (pH, temperature, light, magnetic field).

Example: Poly(N-isopropylacrylamide), poly(acrylic acid).

Application: Targeted drug delivery, water purification.

Z. Biodegradable polymer nanoparticles

Description: Particles that are destroyed by biological factors (enzymes, pH).

Example: Polylactide (PLA), PLGA, poly(hydroxyalkanoates) (PHA).

Application: Biomedicine, ecology.

I. Electrically conductive polymer nanoparticles

Description: Polymer-based particles with conductive properties.

Example: Polypyrrole (PPy), polythiophene.

Application: Power engineering, sensors, electronics.

K. Polymer nanoparticles with functionalized surface

Description: Particles on the surface of which active groups (amino groups, carboxyl groups) are present.

Example: Polystyrene with functional groups.

Application: Selective sorption of molecules, catalytic systems.

Mechanisms of sorption of small molecules

Sorption of small molecules by nanoparticles occurs due to various mechanisms of interaction between the surface of the nanoparticles and the molecules, which can be divided into physical and chemical sorption. Physical sorption is based on weak intermolecular interactions such as van der Waals forces, dipole-dipole interactions and hydrophobic effects. For example, hydrophobic molecules are adsorbed on hydrophobic surfaces such as carbon nanotubes or graphene. Van der Waals forces ensure the attraction of molecules to the surface of nanoparticles due to dipole oscillations. Chemical sorption, on the contrary, involves the formation of strong chemical bonds between molecules and the surface of nanoparticles. It can manifest itself in the form of covalent bonding, ion exchange or coordination bonds. For example, heavy metals can bind to iron oxide, and toxins to molecularly imprinted polymers.

Another important factor is the porosity of the nanoparticles, which ensures size-selectivity of sorption. Porous materials such as zeolites or metal-organic frameworks (MOFs) adsorb small molecules whose sizes correspond to their pore sizes. For example, MOFs effectively sorb gases such as CO₂ or CH₄. Electrostatic interactions also play a significant role: charged surfaces of nanoparticles attract oppositely charged molecules. For example, negatively charged polymer nanoparticles bind positively charged metal ions.

To improve the efficiency of sorption, the surface of nanoparticles can be chemically functionalized, which enhances selectivity for certain molecules. For example, modifying the surface of iron oxide nanoparticles with thiol groups improves their ability to bind mercury. Some nanoparticles have stimulus-sensitive properties: they change their structure or hydrophilicity in response to changes in the environment, such as pH, temperature, or light, which enhances sorption.

Sorption efficiency depends on factors such as the composition of the nanoparticles, their size and surface area, chemical properties of the surface, and the nature of the molecules being sorbed. For example, small particle sizes provide a large surface area, which increases sorption capacity. The presence of functional groups on the surface enhances selectivity for certain molecules.

Sorption of small molecules on nanoparticles can occur both on their surface and inside their volume. These processes depend on the chemical nature of the nanoparticles, their structure and the properties of the target molecules. Surface sorption is the most common process, since nanoparticles have a large specific surface area due to their small size. Small molecules bind to active centers or functional groups located on the surface of nanoparticles. Physical adsorption is based on weak interactions, such as van der Waals forces and dipole-dipole interactions, an example is the adsorption of carbon dioxide on graphene. Chemical adsorption involves the formation of strong chemical bonds between molecules and functional groups on the surface, such as covalent, coordination or ionic bonds. For example, heavy metals bind to functionalized iron oxide nanoparticles. Electrostatic interactions also play a role: charged surfaces of nanoparticles attract oppositely charged molecules or ions, for example, negatively charged nanoparticles bind positively charged cations.

Bulk sorption occurs when nanoparticles are porous or molecules can penetrate into their matrix. This process is especially characteristic of porous materials or nanogels, which are able to "absorb" molecules. In the case of porosity, molecules can penetrate into the pores (for example, in zeolites or metal-organic frameworks), interacting with the inner walls. The size of the pores determines which molecules can be sorbed. Metal-organic frameworks, for example, are actively used to capture gases. In polymer nanoparticles or nanogels, molecules can diffuse into the interior, where they are retained due to interactions with internal functional groups, or are physically "locked" in the structure after changing environmental conditions, such as temperature or pH.

Surface and bulk sorption differ in mechanism, materials, and application. Surface sorption is dominant in compact or nonporous nanoparticles, while bulk sorption is typical for porous materials or gel-like systems. The choice of the appropriate mechanism and type of nanoparticles is determined by the tasks, such as the required selectivity, the rate of the process, or the nature of the molecules being sorbed. Sorption of small molecules is actively used in such areas as water purification, gas capture, targeted drug delivery, and selective filtration.

Reversibility of sorption

In one embodiment of the invention, nanoparticles are selected that are capable of sorbing the said substrate reversibly, for example, due to the presence of desorption, or due to the gradual degradation of particles (including under any conditions or by inducing such degradation by adding chemical/biochemical stimuli for the degradation of a part - certain ions, enzymes (for example, various hydrolases, proteinases, nucleases, etc.).

Desorption of molecules from nanoparticles occurs due to many factors related to physicochemical processes. One of the key factors is adsorption energy. If molecules interact with the surface of a nanoparticle with low energy, for example through weak van der Waals forces, they can easily leave it under the influence of thermal energy. In contrast, at high adsorption energy, as in the case of chemical adsorption, more energy is required for desorption and the process may be less probable. Temperature plays an important role: its increase increases the kinetic energy of molecules, which allows them to overcome energy barriers and desorb.

The composition of the surrounding medium also has a strong influence on the process. The introduction of competing molecules or a change in the chemical composition of the gas or liquid phase can reduce the holding power of the molecules on the nanoparticle surface, stimulating their desorption. In addition, the state of the nanoparticles themselves can change: for example, oxidation, phase transitions, or changes in the surface structure can weaken the interactions between the molecules and the nanoparticle. The nature of the bonds between the molecules and the surface also plays an important role. If these bonds are weak (e.g., dipole-dipole interactions), external influences such as ultrasound or an electric field can destroy them and lead to desorption.

At the nanoscale, due to the high surface area and the peculiarities of the crystalline structure of nanoparticles, their interaction with molecules strongly depends on their size, shape and the presence of defects. For example, different edges or defects can create local areas with different adsorption energies. The moistening or solubility of molecules in the environment also has an effect: if molecules are capable of dissolving, they can go into solution, and condensation of the liquid phase on the surface promotes their desorption. In some cases, desorption is associated with catalytic processes: molecules, interacting with a nanoparticle, change their chemical nature (for example, decompose or oxidize) and leave the surface in the form of new compounds.

Efficient sorption and subsequent desorption of small molecules depends on the properties of the particles, including their material, size, shape, and chemical modification of the surface. Nanoparticles made of metals such as gold, silver, and platinum are widely used due to their high specific surface area, ability to catalytic reactions, and the ability to control interactions with molecules by changing their size and shape. Metal oxide nanoparticles such as titanium dioxide or zinc oxide are also effective because they have active sites for adsorption and can participate in photocatalytic processes, which facilitates desorption. Carbon nanomaterials such as graphene, carbon nanotubes, and porous carbon are characterized by high porosity and large surface areas, which allows them to effectively retain small molecules through physical or chemical adsorption. They are especially effective when desorption is required under the influence of heat or a change in the composition of the medium.

In addition, polymer nanoparticles such as polystyrene or polyacrylamide microspheres can be modified to selectively sorb small molecules and then desorb them when the pH or temperature changes. Hybrid nanoparticles that combine several materials, such as metal-carbon or metal-polymer structures, are used to increase desorption selectivity and efficiency. Nanoparticles with meso- and micropores, such as zeolites or metal-organic frameworks, are capable of not only effectively sorbing small molecules by adsorption inside the pores, but also releasing them when external conditions change, such as pressure, temperature, or gas composition. Such particles are often used in gas storage, catalysis, and purification. It is important that the particles have an optimal size and surface properties, since too small particles can aggregate, reducing their activity, and too large particles can reduce the surface area.

Additional load molecules

In one embodiment of the invention, additional molecules are sorbed into the selected nanoparticle in addition to the substrate - therapeutic, visualizing (various fluorophores, etc.): small molecules, antibiotics, antiviral drugs, analgesics, anti-inflammatory agents, chemotherapeutic agents, enzyme inhibitors, tyrosine kinase inhibitors, hormones, synthetic hormonal analogues, peptides, proteins, therapeutic antibodies, siRNA, antisense oligonucleotides, mRNA, plasmids, recombinant proteins, cytokines, interferons, immunotherapeutic agents, cell therapies, chemical proteases, receptor blockers, radiopharmaceuticals. Therapeutic molecules can be a wide range of compounds that are used in medicine to treat various diseases. In particular, small molecules are chemical compounds with a low molecular weight that easily penetrate cells and interact with proteins, enzymes or receptors. These molecules include analgesics, antibiotics, antivirals, and chemotherapeutics. Then there are biological molecules, which include proteins, peptides, and antibodies. Proteins, such as insulin or erythropoietin, are used to replace or stimulate body functions, while antibodies, such as trastuzumab or adalimumab, selectively bind to specific target proteins to fight cancer or autoimmune diseases.

Peptides occupy an intermediate position between small molecules and proteins and are widely used in therapy, for example, to regulate hormonal functions or control metabolism. Nucleic acids such as siRNA, antisense oligonucleotides and mRNA are a new class of therapeutic agents that interfere with gene expression processes. They are joined by gene therapy agents that deliver genes to correct hereditary defects or treat diseases associated with mutations. Enzyme inhibitors, such as protease inhibitors for the treatment of HIV or tyrosine kinase inhibitors for the fight against cancer, play an important role in blocking key biological processes. Hormones and their synthetic analogues such as cortisol or estrogens regulate the functions of the endocrine system.

Various biopolymers

In one embodiment of the invention, the nanoparticles are additionally conjugated with a polymer from the group: proteins, nucleic acids, polysaccharides, and others. In particular, conjugates were obtained with: immunoglobulins (including IgG, IgM), lectins, peroxidases, luciferases, albumins, DNA, RNA, plasmid DNA, matrix RNA, antisense DNA, antisense RNA, small interfering RNA, piwiRNA, microRNA, aptamer, antisense DNA, dextran, carboxymethyldextran, chitosan, polyethyleneimine, polyethyleneglycol, polyacrylic acid.

In one embodiment of the invention, said protein may be: serum albumin, transferrin, holo-transferrin.

In one embodiment of the invention, said antibody may be a gamma immunoglobulin, immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), immunoglobulin G3 (IgG3), immunoglobulin G4 (IgG4), immunoglobulin M, immunoglobulin A, immunoglobulin E, immunoglobulin D, Fab antibody fragment, Fab' antibody fragment, Fab'2 antibody fragment, F(ab')2 antibody fragment, scFv antibody fragment, nanobody, scFv-Fc fragments, scFv-scFv (diabody), bispecific scFv fragments, Fab-scFv antibody fragment.

In addition, the mentioned antibody is a genetically engineered analogue from the group: avimers, adgyrons, adnectins, monobodies, atrimers, anticalins, affibodies, affilins, affimers, affitins, nanophytins, darpins, knottins, obodis, polypeptides based on the Kunitz domain, pronectins, repbodys, phynomers, centirins, ADAPT (ABD-Derived Affinity Proteins), NanoCLAMP (nano-CLostridial Antibody Mimetic Proteins), ARM (Armadillo repeat proteins), PDZ proteins.

Proteins useful for this aspect of the invention may include, but are not limited to, albumin, transferrin, insulin, endostatin, hemoglobin, myoglobin, lysozyme, immunoglobulins, α-2-macroglobulin protein, fibronectin, laminin, collagen, gelatin, protein components of lipoproteins, synthetic peptides and proteins, and combinations thereof. Examples of suitable proteins include, but are not limited to, albumin, transferrin, insulin, endostatin, hemoglobin, myosin, lysozyme, immunoglobulins, α-macroglobulin, fibronectin, lamin, collagen, Apo-A1, gelatin, artificial proteins, and combinations thereof.

The created conjugates with these polymers showed high specificity of binding to their target (ligand, binding partner).

Microscopic and cytometric analysis

The mentioned cell marker can be any clinically significant marker, in particular, nanoagents have been created that specifically recognize EGFR, HER2, PD-L1, EpCAM, CEA, PSA, CA-125, CA 19-9, CA 15-3, CA 72-4, MUC1, CD19, CD20, CD44, CD133, CD166, CD24, CD47, CD117, DR-5.

Signal registration

The detected signal is recorded qualitatively, semi-quantitatively or quantitatively visually or on various equipment: microscope, cytometer, cytofluorimeter, luminometer.

Cross-linking

In one embodiment of the method, there is an additional step of cross-linking the nanoparticles to stabilize the composition, prevent premature desorption or control the rate of substrate desorption, etc.

for cross-linking of nanoparticles, they are mixed with bifunctional cross-linkers. This can be glutaraldehyde, formaldehyde, diglycidyl ether, genipin, etc. In one embodiment of the method, a cross-linker is used that leaves the particles or makes them highly specific in terms of recognition/binding to their biological target. In particular, cross-linking with formaldehyde or diglycidyl ether is preferable to using glutaraldehyde, since it usually allows for more specific particles. At the same time, for such tasks as non-specific transfection of cells with genetic material, it is preferable to use a cross-linking agent that does not lead to selectivity of particles, but on the contrary allows them to bind highly efficiently to various targets. For example, glutaraldehyde leads to the formation of Schiff bases, which have a partial positive charge, and therefore will effectively perform the task of binding DNA (which is negatively charged) for the purpose of delivering genetic material to cells.

Immobilization of receptors on a solid phase

Methods for immobilizing binding receptors on a membrane are well known. Typically, test and control binding receptors are applied to the membrane in parallel lines at a distance from each other. Solutions with the required substances in appropriate buffers are applied to test strips using automated dispensers. After air drying for the required time, the membrane is blocked in a suitable buffer and stored in a desiccator before assembling the test strip into a single unit.

The speed of analysis using the proposed invention is significantly higher than standard analysis methods while maintaining the accuracy and reliability of the results. In this case, the measurement takes on average from 10 to 300 seconds. However, it is possible to increase the sensitivity if a longer analysis time is used. In addition, the principle of this invention allows for direct measurement of the determined nucleic acid as an aealite without the need for its amplification, provided that its concentration is above the detection limit, and the signal associated with it can be recorded, for example, with the naked eye.

Measurements using the method within the framework of the present invention can be carried out both under stringent and mild conditions. "Stringent" conditions are understood to mean such values of temperature, ionic strength, and the presence of other compounds, at which hybridization of nucleic acids occurs. Under "high stringency" conditions, hybridization will occur only for nucleic acids having a high content of complementary nitrogenous bases. Thus, mild conditions are required in the case when hybridization or annealing of not completely complementary nucleic acids is necessary. A number of methods for creating mild conditions for hybridization are known to those skilled in the art. Hybridization under stringent conditions is possible only in the case of complete complementarity of sequences. Hybridization under less stringent conditions is possible for sequences with less than 100% identity. Changes in environmental conditions, such as a decrease in the salt concentration or an increase in temperature, can make it possible to reduce the stringency of hybridization conditions. Stricter hybridization conditions can be achieved by decreasing the ionic strength or increasing the hybridization temperature.

Thus, in one of the embodiments of the present invention, instead of using the said antibody as a recognition element, it is possible to use DNA/NK receptors. In this case, it is possible to carry out a single-stage, fully functional determination of specific DNA or RNA molecules in an immunochromatographic analysis format, in which all the necessary reagents are on the test strip in anhydrous form. On the other hand, this invention makes it possible to directly determine NK without the need for their amplification and provides a method for their rapid detection.

In addition, in its alternative embodiments, the invention is also applicable for detecting NC under elevated temperature conditions, which expands the scope of its use in various fields, such as forensic examination. In addition to these advantages, the described method is a simple method for detecting nucleic acids and does not require serious training in this field, which can facilitate the penetration of genomic express analysis methods into the field of diagnostics at the patient's bedside and at the point of medical care.

Analysts

The present invention makes it possible to detect a wide range of molecular analytes, including nucleic acids and polynucleotide sequences, including fragments and whole DNA molecules, as well as transport, matrix, ribosomal, micro RNA; synthetic analogues of nucleic acids, such as peptide NA, morpholine, closed, glycolic and threose-NA; and also, due to the use of special recognition oligonucleotides (for example, aptamers) also molecules of non-nucleotide composition, for example, proteins and peptides, hormones, low-molecular physiological regulators, other haptens, metal ions, etc.

Calibration curve

In one embodiment of the method for quantitatively assessing the content of an analyte in a sample (measuring the concentration), an additional step is taken of comparing the obtained results with a calibration curve measured for samples with a known content of the analyte.

Multiplexity

This method is also applicable for the simultaneous determination of several analytes. In the simplest format, the test strip can be divided into separate strips, where separate binding zones are applied for the detection of different analytes. In addition, it is also possible to implement the simultaneous detection of different analytes in one sample. For this purpose, for example, separate binding zones can be applied to one membrane, but in its different areas.

Specific receptor/ligand pairs

Within the framework of the present invention (including instead of the mentioned antibodies), various specific pairs of the "receptor/ligand" type can be used, applicable for creating signal (reporter) conjugates and receptors binding them, as well as recognizing oligonucleotides or protein receptors with their analyte. The components of such pairs applicable in the present invention can be of both immune and non-immune types. Examples of immune binding can be systems based on the interaction of antigen/antibody or hapten/antihapten. Polyclonal, monoclonal antibodies or their immunoactive fragments can be prepared by standard methods known to those skilled in the art. The terms "immunoactive fragment of an antibody" or "immunoactive fragment" refer to parts of antibody molecules that contain a specific binding region. Such fragments may be Fab fragments, which are defined as fragments lacking the Fc portion, such as Fab, Fab', and F(ab)2 fragments, or may be so-called "half-molecular" fragments produced by reductive cleavage of the disulfide bonds linking the heavy chains of the parent antibody. If the antigen in the binding pair is not immunogenic, such as a hapten, it may be covalently linked to an additional carrier protein to render it immunogenic.

Non-immune specific pairs include systems in which two components exhibit a natural affinity for each other without being antibody and antigen. Well-known examples of non-immune pairs include biotin and avidin, biotin and streptavidin, folic acid and folate-binding proteins, complementary nucleic acids, proteins A, G and immunoglobulins, etc. They also include pairs that form covalent bonds with each other: for example, pairs of sulfhydryl groups/maleimides and haloacetyl derivatives) and amino groups/isothiocyanates, succinimide esters and sulfonyl halides, etc. (MN Bobrow, et al., J. Immunol. Methods , 1989,125:279).

Immunochromatographic (ICA) test strips

The ICA test strip is the environment in which the analysis is carried out using the present invention. The test strip typically consists of a chromatographic porous material and includes a sample application zone and a binding zone, as indicated above.

The chromatographic porous materials of which the strip consists are generally hydrophilic and/or can in principle be converted into such, and include the following materials, used either individually or in combination with other materials - granules of inorganic nature, such as silicon, magnesium sulfate, aluminum; natural polymers, in particular cellulose and materials based on it (for example, fibrous polymers - filter paper, chromatography paper, etc.); synthetic or modified natural polymers, such as nitrocellulose, acetylcellulose, polyvinyl chloride, polyacrylamide, cross-linked dextran, agarose, polyacrylates, etc.; ceramic materials; glass; and similar materials. For the described invention, the preferred material is nitrocellulose.

In a preferred embodiment, the membrane may be either a separate structure in the form of a sheet cut into test strips, or may be fixed to a support or solid surface, such as in thin layer chromatography.

Binding of receptors to the membrane surface can be achieved by well-known methods described in the literature. See, for example, Immobilized Enzymes, Inchiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Bio. Chem., 1970, 245:3059.

Preparing the test strip

When preparing a test strip for analysis, the necessary reagents are first applied, and then the test strip is incubated in the necessary buffer solutions.

Buffer solutions and solvents used for the described invention are known (see, for example, Weng et al. (US Patent 4,740,468). As a rule, the pH values of the buffer for conducting an immunochromatographic assay are in the range of 4-11, more often 5-10, for the present invention, 6-9 is preferable. The pH values are selected so as to maintain the necessary level of affinity between the interacting pairs of molecules and to be optimal for signal formation by the corresponding system. Various buffers can be used to achieve the desired pH value and maintain it during the assay. Borate, phosphate, carbonate, Tris, diethylbarbiturate, etc. can be given as an illustration. The use of a specific buffer solution is not significant for the invention as a whole, however, when conducting specific studies, the use of a specific buffer solution may be preferable.

For the analysis within the framework of the present invention, a constant temperature value is usually used. As a rule, for the analysis and obtaining the detectable signal, temperature values in the range of 4-50 °C are used, usually in the range of 10-40 °C, and even more often - temperatures close to the ambient temperature, i.e. 15-25 °C.

When using in one of the embodiments of the present invention, the recognition oligonucleotide and its binding (hybridization) with the target nucleotide sequence requires conditions that facilitate the hybridization of the NC. Hybridization is usually carried out in a Ficoll buffer solution, which may also contain polyvinylpyrrolidine, BSA, yeast tRNA, non-specific DNA, dextran sulfate, dithiothreitol and formamide. Hybridization can occur both in solution and on a solid phase, with one nucleotide chain immobilized on the test strip in the binding zone.

IMMUNOCHROMATOGRAPHIC ANALYSIS

Immunochromatographic assay (ICA) is a rapid and effective method for detecting various biomolecules, such as antigens and antibodies, in biological samples. This method is based on the principle of specific binding of antibodies and antigens and the use of visual changes to determine the presence of target molecules. ICA is widely used in the diagnosis of infectious diseases, health monitoring, and testing the quality of water and food.

Traditional immunochromatographic tests use staining based on chromogenic or colloidal labels, but the use of the ALC according to this invention opens up new possibilities for increasing the sensitivity, accuracy and speed of analysis.

The principle of operation of immunochromatographic analysis using ALC

Immunochromatographic analysis with luminescent nanoparticles uses a principle similar to traditional ICA, but with the addition of a luminescence detection stage. In general, the process can be divided into several stages:

1. Preparation of the test strip: antibodies are fixed on the test strip or membrane, which will bind to the target molecules (antigens or antibodies) present in the analyzed sample. The membrane may also have detection zones where the accumulation of luminescent particles will occur.

2. Incubation with sample and luminescent nanoparticles: the sample (e.g. blood serum, urine, etc.) is added to the test strip. The system is then supplemented with ALP, which can be functionalized with antibodies specific to the target molecules (e.g. antibodies to viruses, bacteria or tumor markers).

3. Reaction development stage: If target molecules (e.g. antigens) are present in the sample, they bind to the antibodies on the test strip and initiate the formation of a complex with the luminescent nanoparticles. These nanoparticles move along the strip until they reach the detection zone.

4. Detection reaction: When the luminescent nanoparticles reach the detection zone, luciferase catalyzes a reaction with its substrate (luciferin), resulting in the release of light (luminescence), the intensity of which will be proportional to the amount of target molecules present in the sample.

5. Measurement and interpretation of the result: Luminescence from particles deposited on the test strip can be measured using specialized equipment (e.g. luminometer or mobile detectors). In some cases, visual assessment is sufficient if an approach is used that allows the luminescence to be observed with the naked eye (e.g. in the case of strong luminescence).

Advantages of using ALC in immunochromatographic analysis

a. High sensitivity and accuracy: luminescent nanoparticles allow for a significant increase in the sensitivity of the analysis compared to traditional methods using chromogenic or colloidal labels. This is especially important for detecting low concentrations of target molecules, such as viral antigens or tumor markers. Luminescence allows for easy detection of even small amounts of molecules in a sample, which significantly increases the accuracy of diagnosis.

b. Minimization of background and interference: The use of ALC reduces the influence of background and other components of the sample that may influence the results of chromogenic reactions. Luminescence is more specific and resistant to interferences (e.g., color or the presence of other substances that may interfere with chromogenic reactions).

c. Speed of analysis: Immunochromatographic tests with luminescent nanoparticles provide faster results, since the luminescence measurement can be performed almost instantly after the addition of the sample and nanoparticles. This allows tests to be performed in a short time, which is especially important for emergency diagnostics in clinical settings.

d. Versatility: ALC can be functionalized to bind to a variety of molecules, allowing the method to be adapted for a variety of diagnostic purposes. This makes it ideal for multi-component tests, such as for the simultaneous detection of different infections or for monitoring multiple biomarkers in a single sample.

d. Convenience and ease of use: immunochromatographic tests with luminescent nanoparticles can be adapted for use in the field and for home diagnostics. Modern portable detectors allow analysis to be carried out directly on site with minimal technical requirements, which makes this method accessible and convenient for a wide range of users.

Application of immunochromatographic analysis with luminescent nanoparticles in biomedical diagnostics

1. Diagnosis of infectious diseases.

Luminescent nanoparticles in immunochromatographic analysis can be used for rapid detection of viral infections such as influenza, COVID-19, hepatitis, as well as for diagnostics of bacterial infections. This can be especially useful in emergency situations, when it is important to quickly make a diagnosis and start treatment.

2. Diagnosis of cardiovascular diseases

The use of luminescent nanoparticles in immunochromatographic analysis allows for the detection of biomarkers associated with various cardiovascular diseases, such as acute myocardial infarction. Rapid detection of markers such as troponin in a patient’s blood can help in prompt diagnosis and initiation of appropriate treatment.

3. Oncology and treatment monitoring

ICA based on ALC can also be used for cancer diagnostics and monitoring by detecting tumor markers in biological fluids and environments. Luminescent nanoparticles specific to certain tumor markers can be used for early diagnostics and disease prognosis assessment.

4. Environmental monitoring

Immunochromatographic analysis with luminescent nanoparticles can also be used for environmental monitoring. This method allows for the rapid detection of pollutants in water, air and soil, as well as for testing food products for toxic substances or pathogens.

Microscopy

To study the interaction of nanoparticles with cells, we used our bioluminescent systems, which allowed us to observe the dynamics of nanoparticle penetration into cells and their localization. Autonomous luminescence provided high image contrast and allowed us to minimize the influence of the background, increasing the resolution and accuracy of observations.

Microscopy

Microscopy is a fundamental tool for imaging cellular structures, molecules, and other biological objects with high resolution. In recent years, with the development of nanotechnology, microscopy has improved significantly through the use of luminescent nanoparticles, which allow for high-contrast, high-precision imaging.

The luminescent particles of the present invention, containing integrated luciferase and its substrate, represent an innovative tool for microscopic research, providing unique opportunities for observing biological processes at the molecular level.

The use of these nanoparticles in microscopy can significantly improve image quality and expand the range of applications, for example in the following areas.

a. Study of cellular interactions and localization of molecules in the cell. Due to their autonomy and high level of luminescence, ALNs allow studying the dynamics of molecular processes in real time and at the level of individual cells, which is impossible using traditional fluorescent markers.

b. Visualization of tumor cells and metastases. For example, the use of ALN functionalized with antibodies against tumor markers allows for precise tracking of tumor growth, their spread, and their impact on surrounding tissues, as well as not only localization of tumor cells, but also assessment of their morphology and response to therapy.

c. Dynamic tracking of cellular processes such as cell migration, cell division, cell death processes (apoptosis, necrosis, etc.), as well as drug interactions in real time and high resolution.

g. Microscopy using multicolor luminescent nanoparticles for multifunctional microscopy. By creating ALN with the ability to emit light in different spectral ranges, it is possible to study several biological objects simultaneously.

At the same time, ALN will provide the following advantages:

1. High sensitivity due to high brightness and contrast of images, which allows to detect even low concentrations of target molecules or cells, inaccessible to traditional methods.

2. Low toxicity and invasiveness due to contactless measurement of results and the absence of the need to introduce a substrate, which will make it possible to use ALN for long-term studies without harm to cells or tissues.

3. High specificity and selectivity when ALN is modified with biomolecules to bind to specific molecular and physiological targets, allowing for precise and highly specific imaging.

4. Durability and stability: The ALNs of the present invention will have high stability and durability, which will allow them to be used as long-lived markers in various microscopic studies.

Cytometry

Nanoparticles were used in flow cytometry and cell sorting. Due to their high sensitivity and autonomous luminescence, our bioluminescent labels allowed us to accurately isolate cells based on their biomarker features, which facilitates the isolation and analysis of rare cells, such as tumor cells circulating in the blood or stem cells.

Cytometry

Cytometry is a technique used to quantify and characterize cells and particles by laser excitation followed by measurement of the light they emit or scatter. Cytometry, especially flow cytometry, is a powerful tool for studying cellular processes such as cell migration, cell death (apoptosis, necrosis, etc.), differentiation, and population changes. The ALPs of the present invention may provide additional advantages for cytometric studies. For example, such particles may be used for the following cytometric applications.

a. Cellular analysis and characterization of cell populations. For example, ALC can be used to label specific cell types (e.g. cancer cells, immune cells, or stem cells) and analyze their population distribution, activity, and other parameters such as cellular maturity or differentiation.

b. Assessment of cell viability and assessment of cell death processes (apoptosis, necrosis, autophagy, etc.). For this purpose, ALC, for example, can be functionalized with receptors specific to molecules activating apoptosis (e.g., caspase proteins).

c. Phenotyping and molecular characterization of cells by interaction of specially functionalized ALCs with specific molecular markers or receptors on the cell surface, which allows for the identification of specific cell populations with high accuracy, as well as the study of their molecular profiles.

g. Real-time monitoring of cellular activity and molecular signals. For example, the use of ALC will allow observation of changes in cellular signaling pathways, such as calcium signals, activation of certain receptors, or the specifics of their interaction with drugs.

For the above cytometric applications, the proposed ALC will have the following advantages.

1. High sensitivity, which will allow detection of even small quantities of cells or molecules. This is especially important for early stage disease diagnostics or for studying rare cell populations.

2. Non-invasiveness. The autonomy of the ALN allows for non-invasive cytometry with minimal impact on living cells without damaging them, which opens up opportunities for long-term research and monitoring of cellular changes.

3. Specificity and selectivity. ALNs can be functionalized to bind to specific cells or molecules, allowing molecular mechanisms to be probed with high selectivity.

4. Multiplexity (simultaneous analysis of multiple parameters) due to the use of luminescent nanoparticles with different properties, which will allow for multiple cell labeling and simultaneous measurement of several parameters (e.g. cell markers, activity labels, etc.).

5. Possibility of quantitative analysis. Cytometry allows not only to visualize cells, but also to conduct their quantitative analysis, which is important for assessing the extent of the disease, the effectiveness of treatment or the impact of biological agents.

Nanothermometry

Our experiments confirmed that bioluminescent nanoparticles have a dependence of the luminescent signal intensity on the ambient temperature. This property makes them promising for nanothermometry, allowing us to measure local temperature changes in tissues or cells. We used these nanoparticles to monitor hyperthermia during therapeutic procedures, which allows us to observe thermal effects and optimize treatment.

Nanothermometry

Nanothermometry is a method of measuring temperature at the nanoscale level, which allows obtaining highly accurate data on temperature changes in local areas such as cells, tissues, molecules or nanoparticles. This technology is widely used in biomedical research to study temperature effects in biological systems such as cell processing, metabolism, response to treatment and other physiological processes. Traditionally, temperature measurement at the microscopic level has been achieved using temperature indicator molecules or nanoparticles that change their properties depending on temperature.

The ALCs of the present invention are a promising tool for nanothermometry, since they have a unique ability to change the intensity or characteristics of their luminescence depending on temperature. This approach can be used for highly sensitive and localized temperature measurement in cells, tissues and other biological objects.

Operating principle

The basic principle of using LCH for nanothermometry is based on the fact that temperature affects the reaction of luciferase with its substrate, as well as the intensity and spectral characteristics of the emitted light.

In particular, LFA can work as temperature indicators. It is important to note that the rate of luciferase reaction and, accordingly, the intensity of luminescence can change depending on both the ambient temperature and the internal environment of the cell. This phenomenon allows these nanoparticles to be used for precise measurement of temperature changes in biological systems.

In general, the measurement process may include the following stages.

1. Labeling with luminescent nanoparticles: ALPs are introduced into the system (e.g. cells or tissues) to be studied. These nanoparticles can be functionalized to bind to specific cells or molecules.

2. Luciferase reaction: During the interaction of nanoparticles with cells or tissues, luciferase catalyzes the reaction of luciferin. Depending on the local temperature, this reaction will occur at different rates, which affects the intensity or spectrum of luminescence.

3. Temperature measurement: Using a detector that analyzes the spectrum and intensity of light, the temperature at the reaction site can be accurately determined. This data can be used to create a map of temperature distributions at the nanoscale.

Application of nanothermometry based on ALC

a. Measuring temperature in cells and tissues

One of the main applications of nanothermometry is the measurement of temperature in cells and tissues, as such data is important for monitoring various biological processes such as metabolism, inflammation, oncogenesis, or response to treatment. For example, in oncology, the use of luminescent nanoparticles to measure temperature in tumor cells can help in studying the effectiveness of thermal ablation of tumors, a method in which tumor tissues are destroyed using high temperatures. By measuring the temperature distribution in tissues, it is possible to precisely control the heating process and avoid damage to healthy tissues.

b. Study of metabolism and cellular processes

Temperature plays an important role in cellular metabolism and molecular processes such as enzymatic reactions, molecular transport, and cellular signaling pathways. Nanothermometry with luminescent nanoparticles can be used to study local temperature changes in cells, allowing us to investigate how temperature changes affect cellular functions, such as heat production by cells during active metabolism or stress conditions (e.g. hypoxia or inflammation).

c. Monitoring temperature changes during exposure to therapeutic agents

The introduction of therapeutic agents such as thermal therapies, drugs or microscopic devices can cause local temperature changes. Nanothermometry allows precise temperature measurement at these locations and monitoring of the effects on tissues or cells, which is important for optimizing therapeutic strategies. For example, in hyperthermia processes for cancer treatment, ALC can be used to monitor the temperature in tumor tissues in real time, allowing for controlled heating of the tumor to the desired temperature and avoiding overheating of healthy tissues.

g. Measuring temperature in biological fluids

Nanothermometry with luminescent nanoparticles can also be used to measure temperature in biological environments and fluids, such as blood, lymph or intercellular fluid. This can be useful for diagnosing various diseases that are accompanied by temperature disturbances in the body, as well as for assessing the effect of drugs on the internal body temperature. For example, studying temperature fluctuations in the blood in response to inflammation or infection can help in the early diagnosis of diseases, as well as in monitoring the body's response to treatment.

Advantages of using ALC in nanothermometry

1. High sensitivity: ALCs have high sensitivity to temperature changes, which allows obtaining accurate data on local temperature changes even at the nano level.

2. Localization and selectivity: Luminescent nanoparticles can be functionalized to bind to specific cells or molecules, allowing for precise temperature measurement in targeted areas, such as tumor cells or during inflammation.

3. Low invasiveness: Nanothermometry using luminescent nanoparticles is non-invasive, which allows monitoring temperature changes in living organisms without damaging them.

4. Real-time data acquisition capability: The use of ALC for nanothermometry enables real-time measurements of temperature changes, which is useful for dynamic monitoring of biological processes.

5. High spatial resolution: Nanothermometry using nanoparticles allows temperature measurements at very small scales (nano level), which is not possible using traditional methods.

Enzyme-linked immunosorbent assay (ELISA)

In our studies, the use of bioluminescent nanoparticles as labels in ELISA showed improved sensitivity and speed of analysis. When bound to target antibodies or antigens, the particles created stable autonomous luminescence, which eliminated the need for adding a substrate. We have successfully applied this technique to the diagnosis of viral infections, hormonal assays, and the detection of autoantibodies.

Enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) is one of the most widely used methods for detecting antigens or antibodies in biological samples. This method is based on the specific binding of antibodies and antigens and allows for the detection of the presence of target molecules with high sensitivity. The standard version of ELISA uses an enzyme as an indicator that catalyzes a reaction with a chromogenic or luminescent substrate, forming a product that can be measured quantitatively.

The integration of luminescent nanoparticles containing luciferase and its substrate into the ELISA process opens up new possibilities for increasing the sensitivity, specificity and versatility of the method. Luminescent nanoparticles allow for faster, more sensitive and highly efficient approaches to detecting target molecules in analyzed samples.

Principle of ELISA using luminescent nanoparticles

In the classical ELISA process, molecules (e.g., antibodies or antigens) are fixed to a solid surface (e.g., in the wells of a microplate) and then react with prototype molecules, such as antibodies or antigens, that are bound to an enzyme. After addition of the substrate, the enzyme catalyzes the formation of a colored or luminescent product. In the case of using the ALS according to the present invention, the assay process includes the following steps.

1. Fixation of antibodies/antigens on the surface: Antibodies or antigens are fixed on the surface of the microplate or other carriers, which can specifically bind to target molecules (e.g. antibodies or antigens in the sample).

2. Introduction of luminescent nanoparticles: ALPs are added to the sample to be analyzed. These nanoparticles can be functionalized with antibodies or other molecules, allowing them to bind to target molecules present in the sample.

3. Luciferase-substrate reaction: When nanoparticles bind to target molecules, luciferase catalyzes the oxidation of luciferin, resulting in the formation of light (luminescence). The intensity of the emitted light is proportional to the amount of target molecules in the sample.

4. Luminescence measurement: The intensity of luminescence is recorded using specialized detectors (e.g., a fluorimeter or spectrophotometer), which allows for quantitative assessment of the content of target molecules (e.g., antibodies or antigens) in the sample.

Advantages of using ALS for ELISA

a. Increased sensitivity: Luminescent nanoparticles have high brightness, which allows for highly sensitive analysis. Unlike traditional chromogenic methods, where color may change at later stages of the reaction, luminescence allows for detection of even small amounts of target molecules. This significantly improves diagnostic capabilities and allows for detection of very low concentrations of antigens or antibodies.

b. Wide dynamic range: luminescent nanoparticles provide a wide measurement range, i.e. the method can be used to analyze both very low and high concentrations of target molecules, which expands the possibilities of using ELISA in various fields of science and medicine.

c. Fast and easy to use: The use of ALC allows for a reduction in the analysis time. Traditional ELISA methods using chromogenic reactions often require a long time to form a distinct color. Luminescent nanoparticles allow for a faster process, as luminescence can be measured more quickly and without the need for additional sample processing.

d. Increased specificity: ALTs can be easily functionalized to bind to specific molecules, increasing the specificity of the assay. This capability allows ELISA to be used for more accurate and selective detection of various biomarkers or pathogens in complex biological matrices.

Application of ELISA based on ALT in biomedical diagnostics

a. Infectious disease diagnostics. ALT in ELISA can be used for rapid and accurate detection of viral and bacterial antigens in patient samples. This approach allows for early diagnosis of diseases and monitoring of infectious processes.

b. Oncological diagnostics. ELISA using luminescent nanoparticles can be used to detect tumor markers in the blood, which allows for early cancer diagnostics and monitoring of patients during treatment. This method allows for tracking changes in the concentration of biomarkers such as CA 125 (ovarian cancer marker), PSA (prostate-specific antigen), and others.

c. Diagnostic automation and multi-component assays. The luminescent nanoparticle ELISA method enables multi-component assays with simultaneous measurement of several target molecules. This is ideal for complex diagnostic panels including assays for several infectious diseases or monitoring of different biomarkers.

Prospects and advantages of the proposed solution :

The development of bioluminescent particles with integrated nanoparticle components has important advantages in areas such as biosensors, cell health monitoring, and nanomedicine, where a high degree of control over biological processes is required.

Bioimaging

Bioimaging is one of the key areas of medicine aimed at visualizing biological processes, structures and functional changes in the body using various technologies. Unlike traditional fluorescence imaging methods, which often face the problem of tissue autofluorescence and high background signal, bioluminescent systems allow achieving a high signal-to-noise ratio due to autonomous emission of light in the visible range, which occurs during the interaction of luciferase and the substrate encapsulated in a nanoparticle.

The ALNs proposed in this invention provide new opportunities for highly sensitive and highly specific bioimaging. These nanoparticles have a unique ability to emit light in response to specific biochemical reactions, which allows them to be used as labels or indicators for visualization and monitoring of various physiological and pathological conditions.

In particular, the ALNs according to this invention can be used for the following areas of bioimaging:

a. Visualization of cellular interactions and molecular processes. For this purpose, ALNs can be further functionalized to bind to specific cellular structures, such as cell membranes, receptors or intracellular components. The luminescent activity generated by the interaction of luciferase with its substrate allows for the visualization of these interactions in real time.

b. Molecular imaging of tumors and their localization. For this purpose, ALNs can be functionalized so that they bind to tumor cells or their markers, such as specific proteins or receptors expressed on the surface of cancer cells.

c. Tissue imaging and disease monitoring. Through special modification, ALNs can acquire the ability to penetrate cell membranes and interact with target molecules, and thus be used for non-invasive monitoring of inflammation, infections, vascular diseases and other pathologies.

g. Intraoperative bioimaging can be useful in surgery where precise localization of tumors or other pathological areas is required during surgery, allowing for precise determination of tumor boundaries and assessment of treatment effectiveness.

The use of ALN for bioimaging will provide the following advantages over traditional methods:

1. High sensitivity due to the integration of luciferase and its substrate, which will allow detection of very small amounts of target molecules or cells for early diagnosis of diseases and monitoring of processes at the cellular level.

2. Low toxicity ALN will be safer for the body, since it allows to avoid the introduction of high concentrations of the substrate, and the amount integrated with the particle will be small and localized directly at the particle.

3. High specificity due to the ability to adjust the properties of particles for high selectivity to specified types of molecular targets, which will significantly increase the accuracy of diagnostics and avoid false results.

4. Low invasiveness, since, once introduced into the body, ALNs are completely autonomous and do not require additional introduction of a substrate, and the measurement itself does not require surgical intervention.

5. Real-time monitoring of biological processes, making ALN an ideal tool for dynamically tracking changes in the body.

ALNs can be introduced into the body in one of the following ways, depending on the specific purpose and the properties of the particles themselves.

Intravenous (IV) administration is one of the most common methods of delivering nanoparticles in vivo and allows for relatively rapid distribution of nanoparticles throughout the body via the circulatory system. The method is particularly useful for studying circulation in the body in real time, for monitoring various physiological processes, and for diagnosing or treating diseases that require rapid distribution of nanoparticles.

Intramuscular (IM) administration is a method of delivering nanoparticles directly into muscle tissue and is suitable for localized research or therapeutic purposes where specific areas of the body need to be targeted, which may be useful for therapeutic intervention, such as treating tumors or inflammation. IM administration is often used to study the interaction of nanoparticles with cells and tissues directly at the site of administration, as well as to create therapeutic drug delivery systems.

Subcutaneous administration (SC) involves the introduction of nanoparticles into the subcutaneous tissue, which allows the nanoparticles to be slowly absorbed into the blood and delivered to various organs in a more dosed and gradual manner, which may be useful for long-term research or therapy, as well as for the creation of chronic disease models.

Intraperitoneal administration (IP) involves the introduction of ALP into the peritoneal cavity, which allows the nanoparticles to be distributed throughout the organs and tissues located in the abdominal cavity. The method is convenient for animal models and allows for the study of the distribution of nanoparticles in various internal organs, such as the liver, kidneys, and intestines, and can therefore be recommended for toxicological studies and for the study of the pharmacokinetics of nanoparticles in living organisms.

Oral administration is a possible method of delivering ALC via the gastrointestinal tract (through the mouth), which is especially important for drug delivery systems intended for use in tablet or capsule form. This method is convenient for patients and animals, as it avoids invasive procedures such as injections. Oral administration can be used to deliver the nanoparticles of the present invention for the purpose of monitoring processes in the gastrointestinal tract, as well as for diagnosing diseases such as infections or digestive diseases.

Nasal and inhalation administration involves the delivery of ALPs through the respiratory tract, allowing them to penetrate the lungs and nasal mucosa. The method allows nanoparticles to be delivered directly to the lungs and other respiratory tract, making it useful for the treatment of respiratory diseases such as asthma or pneumonia, as well as for research into drug delivery to the respiratory system.

For successful use of ALN in vivo, several factors must also be taken into account, such as stability, biocompatibility, and the ability of nanoparticles to penetrate biological barriers and accumulate in target organs. The following approaches, well known in the industry, can be used to improve these parameters.

Functionalization of nanoparticles using various molecules such as polymers, low and high molecular weight ligands, biomolecules, antibodies, etc. to increase their biocompatibility and selectivity;

Stabilization of nanoparticles using various surface coatings consisting of polymers, lipids or other materials to prevent aggregation or degradation of nanoparticles in biological environments;

Implementation of targeted delivery by additionally linking nanoparticles to molecules that have the ability to penetrate specific cell types

We demonstrated the effectiveness of such systems for visualizing the accumulation of nanoparticles in organs after intramuscular administration, which opens up prospects for use in vaccine production. In addition, our studies showed that visualization of bioluminescent nanoparticles in the gastrointestinal tract allows for the diagnosis of gastrointestinal diseases, such as inflammatory processes and ulcerative lesions. Finally, our nanoparticles demonstrated robust accumulation in tumor tissues, which opens the possibility of using them for the diagnosis of oncological diseases, such as breast cancer, with particle surface modification with anti-HER2 antibodies for targeted visualization.

Diagnosis of diseases

Our bioluminescent nanoparticles have demonstrated high efficiency in diagnostics of various diseases. For example, to diagnose oncological diseases, we used particles modified with antibodies against specific tumor markers, including HER2 for breast cancer and PSA for prostate cancer. Nanoparticles bound to tumor cells, which provided autonomous luminescence and allowed accurate visualization and tracking of tumor accumulation in real time. To diagnose infectious diseases, we used nanoparticles functionalized to bind viral antigens, such as SARS-CoV-2 proteins, which allows for rapid and accurate detection of infection.

Express diagnostics of diseases

The use of ALC in disease diagnostics opens up broad prospects due to the high sensitivity, speed and accuracy of obtaining results. These nanoparticles can be used for early detection of diseases, monitoring their progression, and also for assessing the effectiveness of therapy. In combination with existing clinical immunochemical tests, such nanoparticles are a powerful tool for diagnosing many diseases - from infectious diseases to oncological and cardiovascular diseases.

The ALPs of the present invention can be used for rapid and accurate diagnosis of various infectious diseases. These nanoparticles can be functionalized with antibodies or molecules that bind to pathogens - bacteria, viruses or fungi. The use of such nanoparticles in the diagnosis of infections can include the following applications.

a. Rapid diagnostics of viral infections. To diagnose viral diseases such as influenza, COVID-19, hepatitis, HIV and others, luminescent nanoparticles can be used to detect viral antigens in samples of blood, urine, saliva or other biological fluids. Luciferase located on the surface of the nanoparticles catalyzes a reaction with a substrate integrated with the particle, resulting in the release of light, the intensity of which is proportional to the number of viral particles in the sample. The use of this method provides potentially high sensitivity and the ability to early detect viral infections, even in cases where the viral load in the body is still small. The method can be used both for diagnostics in the early stages of the disease and for monitoring the effectiveness of treatment.

b. Rapid diagnostics of bacterial infections such as tuberculosis, pneumonia, genitourinary tract infections and many others. In this case, ALC can be functionalized to bind to receptors (antibodies, haptens, aptamers, etc.) specific to bacterial cells. The advantages of the method include the ability to quickly and sensitively detect bacterial antigens or bacteria in the body, which can lead to an earlier and more accurate diagnosis.

c. Oncological express diagnostics. ALC can act as an important tool in diagnostics and monitoring of various oncological diseases. For example, ALC can be used for early diagnostics of cancer by detecting low levels of tumor markers that are often present in the blood or other biological fluids at the early stages of the disease, but the detection of which by traditional, less sensitive clinical methods is difficult. At the same time, high sensitivity and the ability to detect cancer at the earliest stages, when there are no obvious symptoms yet, makes it possible to begin treatment at earlier stages, which significantly increases the chances of successful recovery. Examples of such markers include prostate-specific antigen (PSA) for detecting prostate cancer, CA125 marker for detecting ovarian cancer, CEA (cancer embryonic antigen) for early diagnostics of colon cancer, and others.

In addition, by conducting real-time analysis, ALC can also be used to monitor disease progression and track possible relapses. They can be used to monitor the dynamics of changes in tumor marker levels during treatment. This approach makes it possible to monitor the effectiveness of therapy in real time, which allows for adjustments to the treatment plan and avoidance of unnecessary or ineffective methods.

g. Luminescent nanoparticles can be used for early diagnostics of cardiovascular diseases, such as acute myocardial infarction, stroke, hypertension and others. This can be achieved by appropriately modifying ALP to provide specific binding to biomarkers that are indicators of inflammation, damage to cardiac muscle cells or vascular dysfunction. An example of such markers are cardiac troponins (I and T) and/or creatine kinase MB, which are present in the blood when the heart muscle is damaged. Due to the high sensitivity of the analysis, it is possible to detect these biomarkers even at low levels in the blood immediately after the complication, which can be useful for diagnosing myocardial infarction at early stages or for monitoring the patient's condition after surgery. In addition, ALP can be used for emergency diagnostics of vascular diseases, such as atherosclerosis, by detecting molecules characteristic of inflammation or plaque formation in the arteries. This achieves non-invasive assessment of the state of blood vessels and the ability to determine signs of early development of atherosclerosis.

To conduct express diagnostic studies using ALC, it is possible to use various formats of analysis implementation, depending on the conditions of use, the type of disease and the requirements for the diagnostic system. Below are examples of various formats that can be used:

1. ALP in the form of a solution or suspension that can be applied to test strips, microchips or used in test tubes. This format is convenient for use in laboratory conditions, where nanoparticles can be quickly mixed with samples (e.g., blood serum or urine) for analysis. This analysis format is characterized by ease of preparation, flexibility in application to various types of samples and the ability to be used in standard laboratory analyzers. The main areas of application are: rapid diagnostics of infectious diseases, cancer, autoimmune diseases, toxic or chemical markers.

2. Immunochromatographic analysis (ICA) using test strips with ALS. In this format, particles can be integrated into the structure of test strips, onto which antigens or antibodies are applied that are capable of binding to target molecules in the patient's sample. In the presence of target molecules (e.g. viral antigens or antibodies), luminescence occurs on the test strip. This format provides portability and ease of use similar to test strips for home tests, no need for additional equipment or qualified personnel, as well as rapid interpretation of results (visual or using a simple detector). An example of this type of application of ALS is the rapid diagnosis of infectious diseases, such as COVID-19, influenza, hepatitis, as well as for testing for allergies or autoimmune diseases.

3. ALCs built into microchips or biosensors. Such analysis formats can be used to detect the luminescent signal emanating from nanoparticles activated by the presence of target molecules after their reaction with the solid phase of the microchip. This type of analysis is characterized by high sensitivity and accuracy, the possibility of automation of analysis and integration with digital systems, as well as obtaining a high degree of multiplexity due to simultaneous analysis for a number of biological targets in one sample. Such systems can be successfully used in modern diagnostic laboratories, mobile diagnostic systems, emergency medical services, and as part of multifunctional devices for diagnosing various diseases.

4. LFA in nanogels or capsules. Autonomous luminescent nanoparticles according to this invention can be enclosed in polymer nanogels or capsules, which allows for controlled release of luciferase and its substrate, as well as for stabilization of the system. These nanogels can be used in the form of injections or implantable systems for monitoring biochemical processes in the body. This format of analysis will allow for the creation of durable and stable formulas for long-term storage.

Can be used for monitoring inside the body, for example for real-time diagnostics.

They can be programmable, with the ability to activate luminescence in response to certain biochemical conditions.

Application: Control of the therapeutic process, monitoring of tumors, inflammations, infections in the body, diagnostics of diseases in dynamics.

5. Luminescent nanoparticles in the form of tablets or powders for direct use

• Description: In this format, nanoparticles can be presented as powder or tablets that are added to samples. They can be activated under certain conditions such as pH change or temperature.

• Advantages:

○ Convenient transportation and storage.

○ Long shelf life.

○ Simple process of use - just dissolve or mix with the sample.

• Application: Rapid diagnostics in resource-limited settings, field use, diagnostics of infectious diseases, monitoring water quality, toxins or allergens.

6. Nanoparticles activated by an external source (e.g. UV radiation)

• Description: Luminescent nanoparticles can be activated by an external light source (e.g. ultraviolet or visible light), resulting in clear imaging results.

• Advantages:

○ High degree of control over the activation process.

○ Possibility of use in portable devices with minimal hardware requirements.

○ Application in various diagnostic points, including field conditions.

• Application: Field diagnostics, emergency diagnostics of infectious diseases, health monitoring in remote areas.

7. Integration with mobile applications or portable detectors

• Description: For ease of use, luminescent nanoparticles can be integrated into devices that are compatible with mobile apps or portable detectors. These devices can measure luminescence levels and transmit the results in digital format for further analysis.

• Advantages:

○ Possibility of use at home.

○ Automatic analysis of results via the application.

○ Portability and convenience.

• Application: Mobile diagnostics, home tests for various infections, allergies, monitoring of chronic diseases, rapid identification of dangerous toxins.

Each of these formats has its own characteristics and can be used in various conditions, from laboratory research to express diagnostics in the field. The choice of the appropriate format depends on the diagnostic purposes, the type of disease, the availability of equipment and the requirements for the speed and accuracy of analysis.

Examples

The present invention will now be described in detail in conjunction with the following examples so that those skilled in the art can better understand the present invention, but the present invention is not limited to the following examples.

Example 1. Synthesis of hybrid nanoparticles based on magnetite with a shell of metal-organic coordination polymers (MOCPs)

The synthesis was carried out according to the protocol described in the article [https://doi.org/10.1016/j.actbio.2019.12.012] with a change in the number of magnetite coatings to obtain a MOF shell. To obtain magnetite (Fe3O4), 0.901 g of urea, 0.15 g of sodium polyacrylate, 0.487 g of _FeCl3 • _6H2O were dissolved in 16 ml of ethylene glycol. The solution was transferred to an autoclave with a Teflon layer and incubated in a drying oven for 12 h at 200 °C. Then it was cooled to room temperature and washed with ethanol and distilled water 3 times by depositing particles on a magnet and replacing the solvent.

For coating particles with a shell of MOF (MIL-100(Fe)) 40.0 mg magnetite and 137 mg Fe(NO₃)₃• 9H₂O was dissolved in 4 ml of distilled water. 20 ml of an aqueous solution containing 68.2 mg of trimesic acid were added to the resulting mixture and left in an ultrasonic bath for 30 min. Then the synthesis was carried out in a microwave reactor for 15 min at 80^OC, after which the obtained particles (Fe3O4@MIL-100(Fe)) were washed with ethanol and distilled water. After coating with a MOF shell, the particles changed color from black to brown.

To stabilize the particles, they were coated with carboxymethyldextran (CMD). For this purpose, 2 ml of an aqueous solution containing 100 mg of CMD sodium salt were added to 5 mg of _Fe3O4 @MIL-100(Fe). The mixture was placed in an ultrasonic bath for 30 minutes, after which it was incubated on a rotator at 30 rpm for 12 hours, then the _Fe3O4 @MIL-100(Fe)@CMD particles were washed 3 times in distilled water.

Example 2. Synthesis of nanoparticles from the polymer of lactic and glycolic acids (PLGA NPs)

The synthesis was carried out according to the protocol described in the article [https://doi.org/10.1021/acsnano.0c03421]. Nanoparticles were obtained by the microemulsion method with subsequent evaporation of the solvent. For this, 300 μl of PLGA at a concentration of 40 g / l in dichloromethane were added to 3 ml of 3% PVA in water. They were treated with ultrasound for 40 sec at a power of 20%. They were evaporated for 1.5 h on a rotator with constant shaking. The particles were washed by three-fold centrifugation at 8500 g for 7 min.

Example 3. Study of the size and morphology of nanoparticles using scanning electron microscopy

Scanning electron microscopy images of Fe ₃ O ₄ @MIL-100(Fe)@CMD, PLGA NPs and Magsphere magnetic carboxypolystyrene nanoparticles (MCPSNPs) were obtained at accelerating voltages of 11, 7 and 11 kV, respectively. Nanoparticle samples in water at a concentration of 1 g/L were air-dried on a silicon substrate on carbon tape and immediately analyzed. SEM images were processed using ImageJ software to obtain particle size distribution. All nanoparticles were shown to be spherical in shape with diameters in the range of 100-130 nm for Fe ₃ O ₄ @MIL-100(Fe)@CMD, 140-500 nm for PLGA NPs and 300-2500 nm for MCPSNPs (Fig. 1).

Example 4. Conjugation of nanoparticles with polymers by the carbodiimide method

Conjugation of nanoparticles with a carboxylated surface with polymers containing amino groups was performed in 2 stages. For activation, 1 mg of nanoparticles was mixed with 5 mg of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 mg of sulfo-N-hydroxysuccinimide (S-NHS) dissolved in 200 μl of 50 mM MES buffer (pH = 4.5). The resulting mixture was sonicated and left to incubate on a rotator at 30 rpm for 20 min at room temperature. The particles were then washed once with phosphate-buffered saline (PBS) and 100 μl of a PBS solution containing 100 μg of polymer (luciferase, polyethyleneimine, streptavidin, antibodies) were added. The particles were washed 3 times in distilled water by magnetic precipitation (for magnetic particles) or centrifugation for 7 min at 8500g (for PLGA NPs).

Example 5. Obtaining autonomous bioluminescent nanoparticles

NanoLuc luciferase was covalently linked to the surface of Fe3O4@MIL-100(Fe)@CMD, PLGA NPs, and carboxypolystyrene magnetic nanoparticles (CPMNPs) using the carbodiimide method as described in Example 3. An autonomous luminescent system was obtained by sorption of furimazine (a substrate for NanoLuc luciferase) onto nanoparticles. For this purpose, 100 μg of particles were incubated with 10 μg of furimazine in 10 μl of distilled water for 10 min, after which the particles were washed 3 times to remove the unsorbed substrate. Furimazine sorption was studied by magnetic separation of particles and their transfer to DMSO for furimazine desorption. After which the percentage of furimazine loading into particles was studied using a calibration curve; it turned out to be from 7% to 30% by weight (with the addition of 0.1 μg to 100 μg of furimazine).

Example 6. Production of autonomous bioluminescent nanoparticles using the biotin-streptavidin system

As an alternative method for obtaining autonomous luminescent nanoparticles, the surface of Fe3O4@MIL-100(Fe)@KMD particles was coated with streptavidin according to the protocol from Example 3. NanoLuc luciferase was pre-conjugated with biotin. For this, 100 μg of luciferase were incubated with 10 μg of NHS-biotin in 100 μl of PBS for 2 h, after which the protein was washed by gel filtration. 100 μg of Fe3O4@MIL-100(Fe)@KMD-streptavidin particles were incubated with 10 μg of furimazine in 10 μl of distilled water for 1 h, after which they were washed 3 times. 100 μg of the resulting particles were incubated with 10 μg of biotinylated luciferase for 5 min, after which they were washed 3 times.

Example 7. Surface modification of bioluminescent nanoparticles

The surface of bioluminescent nanoparticles was additionally modified with functional polymers by the carbodiimide method according to the protocol from Example 3. Fe3O4@MIL-100(Fe)@KMD-NanoLuc was conjugated with antibodies to chloramphenicol (antiCAP), or with branched polyethyleneimine with a molecular weight of 25 kDa (PEI). PLGA NPs were conjugated with antibodies to HER2 receptors (trastuzumab). Sorption of the substrate was carried out after surface modification.

Example 8. Prolonged luminescence of nanoparticles

Fe3O4@MIL-100(Fe)@KMD-NanoLuc nanoparticles were synthesized as in Example 3. 100 μg of nanoparticles with the substrate were transferred to 1 ml of PBS and placed in a dialysis bag with a pore size of 8-12 kDa. The particles were dialyzed against 1 l of PBS. At certain intervals, the luminescent signal of the particles in the dialysis bag was measured using the LumoTrace FLUO biovisualization system (Abisens LLC, Sirius, Russia). Fe3O4@MIL-100(Fe)@KMD-NanoLuc nanoparticles, to which the substrate was added immediately before dialysis, were studied as a control. 24 h after the start of the experiment, a residual signal was detected in the bioluminescent nanoparticles (Fig. 2). The luminescent signal from the control particles could not be detected after 7 hours.

Example 9. Immunochromatographic analysis using autonomous bioluminescent nanoparticles

A solution containing BSA-biotin at a concentration of 3 g/L was applied to the membranes using a spray system at a flow rate of 4 μL/cm and then dried overnight. The test strips were cut using a guillotine cutting module and had a width of 2 mm.

Autonomous bioluminescent particles of Fe3O4@MIL-100(Fe)@CMD-NanoLuc, PLGA-NanoLu, and KPMNP-NanoLu were prepared as in Example 3 and then conjugated with streptavidin. 20 μl of the solution containing 1 μg of particles were applied to the test strips by immersing them in this solution. The strips were analyzed using the LumoTrace FLUO bioimaging system. A luminescent signal was detected at the site of BSA-biotin application (Fig. 3a). Then, an excess of biotin (5 μg) was added to the solution with the particles. A reduced signal was detected on the test strip with Fe3O4@MIL-100(Fe)@CMD-bioNanoLuc compared to the solution without free biotin (Fig. 2b), for the remaining particles the signal was undetectable.

Example 10. Preparation of BSA-CAP conjugate

CAP succinate was dissolved to a concentration of 100 g/L. EDC was dissolved in 50 mM MES, pH6, and then diluted two-fold with DMSO to achieve a final concentration of 100 g/L.
NHS was first dissolved in DMSO and then diluted two-fold with 50 mM MES, pH 6, to achieve a final concentration of 100 g/L.

50 μl of CAP succinate were mixed with 18.3 μl of EDC solution, 13.6 μl of NHS solution, 18.1 μl of a mixture of 50 mM MES, pH 6 and DMSO (1:1 by volume). Activation was carried out for 1 hour at room temperature.

After incubation, 58.5 μl of BSA at a concentration of 100 g/l, 28.7 μl of activated CAP succinate and 42.8 μl of 0.1 M borate buffer, pH 8, were mixed. The tube was centrifuged for 1 min at 10,000 g, and the supernatant was used. The tube was incubated overnight.

To purify the conjugate, it was applied to ZEBA desalting columns (7 kDa MWCO) once according to the manufacturer's recommendations. The conjugate was stored in Milli-Q water.

Example 11. Competitive enzyme immunoassay for the presence of chloramphenicol in a sample using autonomous bioluminescent nanoparticles

Fe3O4@MIL-100(Fe)@KMD-NanoLuc-antiCAP nanoparticles were synthesized as described in Example 6.

For sample analysis, a plate was prepared: 100 μl of BSA-CAP 0.1 g/l in carbonate-bicarbonate buffer (0.1 M, pH 9.6) were placed into the wells of 96-well ELISA plates and incubated at room temperature overnight. The solution was then removed and the wells were washed once with 100 μl of PBS solution (PBS with 0.05% Tween 20). After that, 100 μl of blocking buffer (1% BSA in PBS) were added to the wells. The plate was incubated for 1 h at room temperature and washed three times with PBST.

Next, the sample was directly analyzed: 100 μl of the following mixture was added to the well: 5 μl of Fe3O4@MIL-100(Fe)@KMD-NanoLuc-anti-CAP at a concentration of 2 g/l, 10 μl of CAP-succinate at a given concentration as a sample, 10 μl of 10% BSA and 75 μl of PBS. The plate was incubated for 1 h, washed three times with PBST, and PBS was added to the wells. The resulting luminescent signal was measured using a 480/80 nm emission filter. An increase in the luminescence signal was observed with a decrease in the analyte concentration with a detection threshold of 0.5 mg/l (Fig. 4).

Example 12. Cultivation of cell lines

The experiments were carried out using EMT 6/P and EMT-HER2 cell lines (a cell line stably expressing the HER2 transmembrane receptor, obtained from the EMT6/P line by lentiviral transduction, followed by selection of HER2-positive clones).

The cells were cultured under the following conditions: modified Eagle Medium from Dulbecco/basic F12 medium (1:1) from Ham; 100 U/ml penicillin-streptomycin; 10 mg/l gentamicin; 10% fetal bovine serum; 300 mg/l L-glutamine; 5% CO2 at 37 °C. Cells were removed from the surface of the culture flask using Versene solution.

Example 13. Carrying out microscopic analysis of a cell sample

20,000 EMT and EMT-HER2 cells were seeded in 96-well plates in 200 μl of medium and incubated overnight. Then, 10 μl of Fe3O4@MIL-100(Fe)@KMD-NanoLuc particles prepared as described in Example 5 at a concentration of 0.1 g/l in PBS were added to the cells. A permanent magnet was applied to the bottom of the culture plate for 1 min. Microscopic images of the cells were then obtained in the bright field channel and in the absence of light sources for detecting the luminescent signal (Fig. 5).

Example 14. Cytometric analysis of a cellular sample using autonomous bioluminescent nanoparticles Fe3O4@MIL-100(Fe)@KMD-NanoLuc-PEI

200,000 EMT 6/P cells in 200 μl of 1% BSA in PBS were mixed with 5 μg of Fe3O4@MIL-100(Fe)@CMD-NanoLuc-PEI particles prepared as described in Example 6. The resulting mixture was incubated for 20 min, centrifuged at 300 g for 3 min and resuspended in 100 μl of PBS. The cells were then immediately analyzed by flow cytometry. A peak shift was observed in the 530-30 emission channel excited by a 488 nm laser compared to the non-luminescent particles (Fig. 6).

Example 15. Specific interaction of nanoparticles with cells

100,000 EMT6/P and EMT-HER2 cells in 100 μl of 1% BSA in PBS were mixed with 1 μg of PLGA@NanoLuc@trastuzumab particles obtained as described in Example 6. The resulting mixture was incubated for 20 min, centrifuged at 300 g for 3 min and resuspended in 100 μl of PBS. The signal was analyzed using a multimodal plate reader in luminescence mode with an emission filter of 480/80 nm. On EMT-HER2 cells, an increase in the luminescent signal was observed with PLGA-NanoLuc-trastuzumab particles compared to PLGA-NanoLuc particles. However, on HER2-negative EMT6/P cells, no statistically significant difference in the luminescence of the particles was observed (Fig. 7).

Example 16. Ex vivo luminescent bioimaging and biodistribution

BALB/c mice were retro-orbitally injected with 300 μg of KPMNPs-NanoLuc, Fe3O4@MIL-100(Fe)@KMD-NanoLuc and PLGA-NanoLuc NPs in 100 μl PBS. Then, 1 hour after the injection, the mice were anesthetized and euthanized by cervical dislocation, and the organs (liver, lungs, kidneys, spleen, heart) were removed and visualized using the LumoTrace FLUO bioimaging system without an emission filter. The resulting images in the bright field channel and in the fluorescence mode are shown in Fig. 8. The most intense signals from the nanoparticles were observed in the spleen, lungs and liver. The distribution of magnetic particles measured by the fluorescence method coincided with the distribution obtained by the MPQ method.

Example 17. In vivo luminescent bioimaging

Pre-shaved BALB/c mice were retro-orbitally injected with 300 μg Fe3O4@MIL-100(Fe)@KMD-NanoLuc in 100 μl PBS. Immediately after nanoparticle administration, mice were placed in the LumoTrace FLUO bioimaging system. Nanoparticle luminescence kinetics were recorded for 30 min. After 15 seconds of circulation, a signal was detected in the lungs, and after 1 min - in the spleen (Fig. 9).

Example 18. Preparation of autonomous bioluminescent nanoparticles with Firefly, Renilla and Gaussia luciferases

Nanoparticles Fe3O4@MIL-100(Fe)@KMD were conjugated with Firefly, Renilla and Gaussia luciferases as in Example 4. Particles with Firefly were adsorbed with D-luciferin (with or without) ATP, and particles with Renilla and Gaussia were adsorbed with coelenterazine (coelenterazine) as in Example 5. Bioluminescent particles with Renilla and Gaussia and Firefly (in the case of ATP adsorption) demonstrated autonomous luminescence in various model environments, particles with Firefly but without ATP - only in the case of the presence of ATP in the environment.

Example 19. Specific interaction of autonomous bioluminescent nanoparticles with modified surfaces with various molecular targets

Nanoparticles Fe3O4@MIL-100(Fe)@KMD-NanoLuc was further conjugated with various types of targeting molecules, including anti-CD4 Fab fragment, anti-CD3 F(ab')2 fragment, anti-CD8 scFv fragment, anti-HER2 affibody and DARPin G3. The substrate was adsorbed after surface modification as in Example 5. The modified particles were incubated with various cell lines overexpressing certain surface receptors and analyzed by flow cytometry as in Example 14. A statistically significant shift of the fluorescence peak to the region of higher intensities was observed upon interaction of CD3+ cells with particles containing anti-CD3 F(ab')2, CD4+ cells with particles containing anti-CD4 Fab, CD8+ cells with particles containing anti-CD8 scFv fragment, HER2+ cells with particles containing anti-HER2 affibody and DARPin G3.

Example 20. Loading of small therapeutic molecules into luminescent nanoparticles

Nanoparticles modified with streptavidin were obtained as in Example 4. After sorption of furimazine, low-molecular therapeutic molecules were similarly sorbed into the particles, including the chemotherapeutic agents doxorubicin and/or daunorubicin, after which the particles were washed from unsorbed molecules. Simultaneous sorption of the substrate and the therapeutic agent was observed. Then, luminescent particles were obtained as in Example 6. Prolonged luminescence of the obtained particles was observed as in Example 8.

Example 21. Use of bioluminescent nanoparticles for theranostic purposes

Autonomous bioluminescent nanoparticles based on Fe3O4@MIL-100(Fe)@KMD and non-magnetic MIL-100(Fe)@KMD with an additional therapeutic load in the form of the chemotherapeutic drug doxorubicin were obtained as in Example 20. 300 μg of nanoparticles with doxorubicin in 100 μl of PBS were retroorbitally administered to mice with pre-inoculated tumors. A luminescent signal in the tumors and a detectable decrease in tumor size were observed compared to intact animals. To improve the therapeutic potential of the nanoparticles, the technology of cytoblockade of the mononuclear phagocyte system was used. For this purpose, 8 hours before the introduction of the nanoparticles, the mice were administered 25 μg of anti-erythrocyte antibodies Ter-119. Using the proposed technology, a more pronounced therapeutic effect of nanoparticles was observed. Additionally, a magnet was applied to the tumor grafting site, which allowed for a significant increase in the accumulation of magnetic nanoparticles in the tumor, as well as a statistically significant extension of the lifespan of mice compared to non-magnetic particles. A decrease in the toxicity of the therapy was also observed compared to free doxorubicin (administered to control mice in the same total dose) according to biochemical indicators.

Example 23. Loading of nucleic acids into luminescent nanoparticles

Autonomous luminescent nanoparticles modified with polyethyleneimine were obtained as in Example 7. Then, 100 μg of nanoparticles were incubated with 10 μg of nucleic acid (plasmid DNA or matrix RNA) in 1 ml of nuclease-free water for 10 min, after which unbound nucleic acid molecules were washed off. Immobilization of nucleic acids on the surface of nanoparticles was confirmed using PCR. Prolonged luminescence of nanoparticles was observed as in Example 8, and luminescent visualization of cells was demonstrated as in Example 13, and simultaneous transfection using reporter systems. Transfection using the obtained nanoparticles and mRNA or plasmid DNA was observed due to expression of the transgene - green fluorescent protein encoded in nucleic acid sequences.

Example 24. Direct enzyme immunoassay for the presence of troponin in a sample using autonomous bioluminescent nanoparticles

Autonomous bioluminescent nanoparticles with a surface modified with antibodies to troponin (clone 9G6 or polyclonal antibodies) were obtained as in Example 6. Antibodies to troponin (clone 1C11cc or polyclonal antibodies) were immobilized on the bottom of the plate as in Example 11. Enzyme-linked immunosorbent assay for the presence of troponin was performed in a sandwich format. A solution with different concentrations of troponin was added to the wells of the plate, incubated for 1 h and washed with a PBS solution. Then 100 μl of a nanoparticle solution at a concentration of 0.1 g/l were added. The plate was incubated for 1 h, washed three times with PBST, and PBS was added to the wells. The resulting luminescent signal was measured using a 480/80 nm emission filter. An increase in the luminescence signal was observed with increasing analyte concentration with a detection threshold of 0.13 ng/ml.

Example 25. Obtaining autonomous far-wave bioluminescent nanoparticles with NanoLuc luciferase

Luminescent nanoparticles containing NanoLuc luciferase were conjugated with various fluorescent proteins, including CyOFP1, TurboGFP, and LSSmScarlet as in Example 4, and then furimazine was adsorbed as in Example 5. The resulting autonomous bioluminescent systems exhibited a shift in the emission spectrum to a longer wavelength region, with a maximum at a wavelength of 502 nm for TurboGFP, 590 nm for CyOFP1, and 598 nm for LSSmScarlet in the absence of an external light source.

Example 26. Obtaining autonomous far-wave bioluminescent nanoparticles with Firefly luciferase

Luminescent nanoparticles containing Firefly luciferase were conjugated with mScarlet, RFP611, and RFP618 fluorescent proteins as in Example 4, after which D-luciferin was adsorbed as in Example 5. The resulting autonomous bioluminescent systems exhibited a shift in the emission spectrum to a longer wavelength region, with a maximum at a wavelength of 594 nm for mScarlet, 611 nm for RFP611, and 618 nm for RFP618 in the absence of an external light source.

The essence of the invention is explained by the drawings Figs. 1-9.

Fig. 1 shows scanning electron microscope images of (A) MCNPs, (B) Fe3O4@MIL-100(Fe)@KMD, (C) PLGA NPs. The scale bar is 500 nm. All nanoparticles are spherical. The diameter of MCNPs, according to this photograph, ranges from 300 to 2500 nm; Fe3O4@MIL-100(Fe)@KMD – from 100 to 130 nm, and PLGA NPs – from 140 to 500 nm.

Fig. 2 shows the luminescence kinetics of Fe3O4@MIL-100(Fe)@KMD-NanoLuc nanoparticles in a dialysis chamber permeable to furimazine. The upper curve corresponds to bioluminescent nanoparticles with pre-sorbed substrate for 10 min. The lower curve corresponds to particles to which the substrate was added immediately before dialysis. Particles with pre-sorbed substrate demonstrate more than 3 times longer luminescence compared to the control.

Fig. 3 shows the results of immunochromatographic analysis (A) in the absence and (B) in the presence of the analyte (biotin) in the sample. BSA-biotin is coated on the test strips. The photographs show the test strips interacted with (left to right) Fe3O4@MIL-100(Fe)@KMD-NanoLuc-streptavidin, KPMNP-NanoLuc-streptavidin and PLGA NP-NanoLuc-streptavidin. The photographs were obtained using the LumoTrace FLUO bioimaging system. The upper panel shows images in a bright field, and the lower panel shows images without external illumination. The places where BSA-biotin is applied to the strips are circled in the photographs. Strong binding of particles that do not interact with biotin in solution to the test line is observed on the test strips. Binding of particles pre-mixed with excess biotin is significantly weaker.

Fig. 4 shows the results of competitive enzyme immunoassay of the sample for the presence of CAP using bioluminescent nanoparticles Fe3O4@MIL-100(Fe)@KMD-NanoLuc-antiCAP. The detection limit using this system is 5 mg/l.

Figure 5 shows microscopic photographs of cell samples interacting with bioluminescent Fe3O4@MIL-100(Fe)@KMD-NanoLuc nanoparticles under an applied external magnetic field. The photographs were taken using objectives with a magnification of (A) x20 and (B) x40. The scale bar is 100 μm. The upper panel shows photographs in a bright field, and the lower panel shows photographs in the absence of illumination. A luminescent signal is observed from cells interacting with the particles (right), while luminescence from intact cells (left) is absent.

Figure 6 shows the results of cytometric analysis of the interaction of EMT6/P cells with bioluminescent Fe3O4@MIL-100(Fe)@KMD-NanoLuc-PEI nanoparticles. (A-B) Scheme of cytometric event selection: first, events with too low forward scattering value are cut off (A), and then events with too high FSC-A parameter value for a given FSC-H value (B). (C) Histograms of intensity in the FITC-H fluorescent channel for cells interacted with luminescent nanoparticles and particles without substrate. A peak shift is observed due to nanoparticle luminescence.

Fig. 7 shows the results of the test for the specificity of the interaction of targeted bioluminescent particles PLGA NP-NanoLuc-trastuzumab with target cells. On EMT-HER2 cells, an increase in the luminescent signal with PLGA-NanoLuc-trastuzumab particles is observed compared to PLGA-NanoLuc particles. At the same time, on HER2-negative EMT6/P cells, there is no statistically significant difference in the luminescence of the particles.

Fig. 8 shows ex vivo biodistributions of (A) KPMNPs-NanoLuc, (B) Fe3O4@MIL-100(Fe)@KMD-NanoLuc and (C) PLGA-NanoLuc NPs. The upper panel contains bright field photographs, the middle panel contains photographs without external illumination, and the lower panel contains diagrams of the distribution of nanoparticles in organs obtained from the analysis of the luminescent signal. It is seen that KPMNPs-NanoLuc and Fe3O4@MIL-100(Fe)@KMD-NanoLuc accumulate predominantly in the lungs and spleen, and PLGA-NanoLuc NPs accumulate in the spleen, liver and lungs.

Fig. 9 shows the in vivo biodistribution of bioluminescent Fe3O4@MIL-100(Fe)@KMD-NanoLuc nanoparticles in the BALB/c mouse. (A) Bright field photograph. (B-D) Photographs obtained in the absence of external light sources with exposures of (B) 25 sec, (C) 2 min, and (D) 15 min. Accumulation of nanoparticles is detected first in the lungs and then in the spleen.

Claims (20)

1. A method of optically controlled theranostics, which includes the following steps: i) select the luciferase enzyme and its substrate, ii) select a therapeutic molecule, iii) selecting nanoparticles capable of sorbing said substrate and said therapeutic molecule, iv) mixing and incubating said nanoparticles with a solution of said substrate of said luciferase to sorb the substrate into the nanoparticle, v) mixing and incubating said nanoparticles with a solution of said therapeutic molecule for its sorption into the nanoparticle, vi) conjugating said nanoparticles with said luciferase enzyme, vii) washing off said substrate that is not bound to said nanoparticle and obtaining theranostic autonomously bioluminescent nanoparticles, viii) induce cytoblockade of the macrophage phagocytic system in the body by introducing antibodies against the erythrocytes of the said organism, ix) introduce into the body the mentioned theranostic autonomously bioluminescent nanoparticles, x) detecting a bioluminescent signal from said autonomously bioluminescent nanoparticles to optically monitor the localization of delivery of said nanoparticles carrying said therapeutic molecule in the body. 2. The method according to claim 1, wherein said nanoparticles are pre-functionalized with a first binding partner, luciferase is functionalized with a second binding partner, and the process of conjugation of the nanoparticle with luciferase is realized due to the interaction of the first and second binding partners and the formation of a bond between them, wherein said first and second binding partners constitute a pair of biotin and streptavidin. 3. The method according to claim 1, wherein said step of mixing and incubating said nanoparticles with a solution of said substrate is performed before said step of conjugating said nanoparticles with said luciferase enzyme. 4. The method according to item 1, in which nanoparticles are selected that are capable of sorbing the substrate of the said luciferase with a substrate loading by weight of more than 1% of the nanoparticle weight. 5. The method according to claim 1, wherein said nanoparticles are polymeric nanoparticles. 6. The method according to claim 1, wherein said nanoparticles are metal-organic framework polymer particles. 7. The method according to claim 1, wherein said nanoparticles are nanoparticles based on a copolymer of lactic and glycolic acids. 8. The method according to claim 1, wherein said luciferase is selected from the series: nanoLuc luciferase, firefly luciferase, thermostable luciferase, Renilla luciferase, Gaussia luciferase. 9. The method according to claim 1, wherein said nanoparticle is conjugated with a receptor against a molecular target selected from the group: gamma-immunoglobulin, immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), immunoglobulin G3 (IgG3), immunoglobulin G4 (IgG4), immunoglobulin M, immunoglobulin A, immunoglobulin E, immunoglobulin D, Fab-fragment of an antibody, Fab'-fragment of an antibody, Fab'2-fragment of an antibody, F(ab')2-fragment of an antibody, scFv-fragment of an antibody, nanobodies, scFv-Fc fragments, scFv-scFv (diabodies), bispecific scFv fragments, Fab-scFv-fragment of an antibody, avimers, adgyrons, adnectins, monobodies, atrimeters, anticalins, affibodies, affililines, affimers, affitins, nanophytins, darpins, knottins, obodis, Kunitz domain-based polypeptides, pronectins, repbodys, phynomers, centirins, ADAPT (ABD-Derived Affinity Proteins), NanoCLAMP (nano-CLostridial Antibody Mimetic Proteins), ARM (Armadillo repeat proteins), PDZ proteins. 10. The method according to claim 1, wherein said therapeutic molecule is selected from the series: an antibiotic, an antiviral drug, an analgesic, an anti-inflammatory agent, a chemotherapeutic agent, an enzyme inhibitor, a tyrosine kinase inhibitor, a hormone, a synthetic hormonal analogue, a peptide, a protein, a therapeutic antibody, a recombinant protein, a cytokine, an interferon, an immunotherapeutic agent, a chemical protease, a receptor blocker, a radiopharmaceutical, DNA, RNA, plasmid DNA, messenger RNA, antisense DNA, antisense RNA, small interfering RNA, piwiRNA, microRNA, aptamer.