WO2019014389A1

WO2019014389A1 - Stimuli-responsive polysaccharide antimicrobial agents

Info

Publication number: WO2019014389A1
Application number: PCT/US2018/041703
Authority: WO
Inventors: Young Jik KWON; Julius Edson
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2017-07-11
Filing date: 2018-07-11
Publication date: 2019-01-17
Anticipated expiration: 2020-01-11

Abstract

The disclosure provides for stimuli-responsive polysaccharide antimicrobial agents, their use as broad-spectrum antibiotics.

Description

STIMULI-RESPONSIVE POLYSACCHARIDE

ANTIMICROBIAL AGENTS

CROSS REFERENCE TO RELATED APPLICATIONS

[ 0001 ] This application claims priority under 35 U.S.C. §119 from Provisional

Application Serial No. 62/531,333, filed July 11, 2017, the disclosures of which are incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

[ 0002 ] This invention was made with Government support under Grant No. DGE-

1321846, awarded by the National Science Foundation. The Government has certain rights in the invention.

TECHNICAL FIELD

[ 0003] The disclosure provides for stimuli-responsive polysaccharide antimicrobial agents, and their use as broad-spectrum antibiotics.

BACKGROUND

[ 0004 ] Chitosan is an abundant antimicrobial agent that is biodegradable with no reported toxicity at low concentrations. At therapeutic concentrations of the free polymer, however, the lack of solubility of chitosan under physiological conditions and material precipitation, leads to toxicity in vitro and in vivo.

SUMMARY

[ 0005] The disclosure provides for stimuli-responsive polysaccharide antimicrobial agents comprising acid-transforming chitosan (ATC). The antimicrobial agents of the disclosure are biodegradable and exhibit antimicrobial activity across a wide pH range. Additionally, the antimicrobial agents of the disclosure exhibit one or more of the following properties: in a neutral aqueous environment, the antimicrobial agents of the disclosure are water soluble when used up to a certain concentration; when used at higher concentrations in a neutral aqueous environment, the antimicrobial agents of the disclosure form a suspension; and when used in an acidic aqueous environment, the antimicrobial agents of the disclosure hydrolyze to chitosan. In comparison to chitosan, the antimicrobial agents of the disclosure exhibit significantly lower toxicities when used at high concentrations under physiological concentrations, while still exhibiting antimicrobial activity.

[ 0006] In furtherance of any embodiment presented herein, the disclosure provides for a stimuli-responsive polysaccharide antimicrobial agent that comprises, consists essentially of, or consists of the structure of Formula I:

Formula I

or a pharmaceutically acceptable salt thereof, wherein, AR¹ is an acid labile group that can be cleaved or hydrolyzed when exposed to a mildly acidic environment (e.g., an environment that has a pH from 4 to 6.8); AR² is an acid labile group that can be cleaved or hydrolyzed when exposed to a mildly acidic environment (e.g., an environment that has a pH from 4 to 6.8); and n is an integer greater than 10. In furtherance of any embodiment presented herein,,

AR² is selected

each independently selected from H, -CH₃, -CH2CH3, -OCH3, and -OCH2CH3, an optionally substituted (Ci- Ci2)alkyl group, an optionally substituted (Ci-Ci2)heteroalkyl group, an optionally substituted (Ci-Ci2)alkenyl group, an optionally substituted (Ci-Ci2)heteroalkenyl group, an optionally substituted (Ci-Ci2)alkynyl group, an optionally substituted (Ci-Ci2)heteroalkynyl group, an optionally substituted (C4-C8)cylcoalkyl group, an optionally substituted aryl group, or an optionally substituted heterocycle, wherein R² and R³ may be connected to each other to form a ring structure, and wherein R⁷ and R⁸ may be connected to form a ring structure. In furtherance of any embodiment presented above, the stimuli-responsive polysaccharide antimicrobial agent comprises, consists essentially of, or consists of the structure of Formula

1(a):

Formula 1(a)

or a pharmaceutically acceptable salt thereof,

wherein, R¹ is a substituted (Ci-Ci2)alkyl group, a substituted (Ci-Ci2)heteroalkyl group, a substituted (Ci-Ci2)alkenyl group, a substituted (Ci-Ci2)heteroalkenyl group, a substituted (Ci-Ci2)alkynyl group, a substituted (Ci-Ci2)heteroalkynyl group, a substituted (C₄- C8)cylcoalkyl group, a substituted aryl group, or an optionally substituted heterocycle, wherein R¹ comprises at least a substitution or group that can be protonated in an acidic environment; R⁶ is a substituted (Ci-Ci2)alkyl group, a substituted (Ci-Ci2)heteroalkyl group, a substituted (Ci-Ci2)alkenyl group, a substituted (Ci-Ci2)heteroalkenyl group, a substituted (Ci-Ci2)alkynyl group, a substituted (Ci-Ci2)heteroalkynyl group, a substituted (C₄- C8)cylcoalkyl group, a substituted aryl group, or an optionally substituted heterocycle, wherein R⁶ comprises at least a substitution or group that can be protonated in an acidic environment; R², R³, R⁷, and R⁸ are each independently selected from H, -CH3, -CH2CH3, - 2 and R³ may be connected together to form a ring structure

⁷ and R⁸ may be connected together to

form a ring structure

and n is an integer greater than

100. In furtherance of any embodiment presented above, the stimuli-responsive

polysaccharide antimicrobial agent of claim 3, wherein R¹ and R⁶ is substituted with an amino group or a sulfhydryl group. In furtherance of any embodiment presented above, the stimuli-responsive polysaccharide antimicrobial agent comprises, consists essentially of, or consists of the structure of Formula 1(b):

Formula 1(b)

or a pharmaceutically acceptable salt thereof,

wherein, R², R³, R⁷, and R⁸ are each independently selected from H, -CH3, -CH2CH3, -OCH3, or -OCH CH wherein R² and R³ ma be connected together to form a ring structure selected

from nd R⁸ may be connected together to form

struct

ure selected from or and n is an integer greater than 10. In furtherance of any embodiment presented above, stimuli-responsive polysaccharide antimicrobial agent of claim 6, wherein the stimuli-responsive polysaccharide antimicrobial agent comprises, consists essentially of, or consists of the structure of Formula 1(c):

Formula 1(c)

or a pharmaceutically acceptable salt thereof, wherein n is an integer greater than 10. In furtherance of any embodiment presented above, the stimuli-responsive polysaccharide antimicrobial agent exhibits the following properties: in a neutral aqueous environment, the stimuli-responsive polysaccharide antimicrobial agent is generally water soluble; and in a mildly acidic aqueous environment, the stimuli-responsive polysaccharide antimicrobial agent is hydrolyzed to chitosan. In furtherance of any embodiment presented above, the disclosure also provides for a composition comprising, consisting essentially of, or consisting of the stimuli-responsive polysaccharide antimicrobial agent described above that is complexed with or polyplexed with one or more nucleic acids, antibiotics, proteins, and/or antifungal agents. In furtherance of any embodiment presented above, the one or more nucleic acids are deoxyribonucleic acid (DNA), polydeoxyribonucleotide (pdrn) and/or complementary DNA (cDNA). In furtherance of any embodiment presented above, the one or more nucleic acids are ribonucleic acids (RNAs). In furtherance of any embodiment presented above, the one or more nucleic acids are small interfering RNAs (siRNAs), CRISPR RNAs, small non-coding microRNAs (miRNAs), and/or antisense RNAs (asRNAs). In furtherance of any embodiment presented above, the siRNAs, CRISPR RNAs, miRNAs or asRNAs target genes necessary for fungal or bacterial growth or replication, target genes for antibiotic resistance, target host genes that are involved in the immune response caused by a fungal or bacterial infection, and/or target the host genes involved in mediating the entry of bacteria or fungi into host cells. In furtherance of any embodiment presented above, the stimuli-responsive polysaccharide antimicrobial agent is complexed or polyplexed with a CRISPR-Cas system, a CRISPRi system, a CRISPR-Cpfl system that targets microbial genes responsible for antibiotic or antifungal resistance, targets microbial genes for microbe growth and survival, or targets microbial genes that result in the death of the microbe. In furtherance of any embodiment presented above, the one or more antibiotics is selected from Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Streptomycin, Spectinomycin, Geldanamycin, Herbimycin, Rifaximin, Loracarbef,

Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin,

Cephradine, Cephapirin, Cephalothin, Cefalexin, Cefaclor, Cefoxitin, Cefotetan,

Cefamandole, Cefmetazole, Cefonicid, Loracarbef, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Moxalactam, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Dalbavancin, Oritavancin, Clindamycin, Lincomycin, Daptomycin, Azithromycin, Clarithromycin, Roxithromycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Azlocillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin, Ticarcillin, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, Ticarcillin/clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nadifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole,

Trimethoprim-Sulfamethoxazole, Sulfonamidochrysoidine, Demeclocycline, Doxycycline, Metacycline, Minocycline, Oxy tetracycline, Tetracycline, Clofazimine, Dapsone,

Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline, Tinidazole, and Trimethoprim. In furtherance of any embodiment presented above, the one or more antifungal agents is selected from itraconazole, posaconazole, ketoconazole, clotrimazole, miconazole, voriconazole, caspofungin, anidulafungin, micafungin, nystatin, amphotericin b, griseofulvin, terbinafine, and/or flucytosine. In furtherance of any embodiment presented above, the stimuli-responsive polysaccharide antimicrobial agent is complexed with or polyplexed with pdrn so as to form particles having diameters greater than 10 nm. In furtherance of any embodiment presented above, the particles have diameters of around 200 nm. In furtherance of any embodiment presented above, the polyplexes have a N/P ratio of about 100. In furtherance of any embodiment presented above, the particles are highly monodisperse particles with a polydispersity index of less than 0.1. In furtherance of any embodiment presented above, the disclosure also provides for a pharmaceutical composition that comprises a stimuli-responsive

polysaccharide antimicrobial agent described above and a pharmaceutically acceptable carrier. In furtherance of any embodiment presented above, the pharmaceutical composition is formulated for oral, parenteral or topical administration. In furtherance of any embodiment presented above, the pharmaceutical composition if formulated for parenteral administration and comprises up to 200 mg/kg of the stimuli-responsive polysaccharide antimicrobial agent. In furtherance of any embodiment presented above, the pharmaceutical composition further comprises one or more additional therapeutic agents. In furtherance of any embodiment presented above, the one or more additional therapeutic agents are one or more antibiotics. In furtherance of any embodiment presented above, the one or more antibiotics is selected from Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Streptomycin, Spectinomycin, Geldanamycin, Herbimycin, Rifaximin, Loracarbef,

Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin,

Cephradine, Cephapirin, Cephalothin, Cefalexin, Cefaclor, Cefoxitin, Cefotetan,

Cefamandole, Cefmetazole, Cefonicid, Loracarbef, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Moxalactam, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Dalbavancin, Oritavancin, Clindamycin, Lincomycin, Daptomycin, Azithromycin, Clarithromycin, Roxithromycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Azlocillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin, Ticarcillin, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam,

Ticarcillin/clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nadifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole,

Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline, Tinidazole, and Trimethoprim. In furtherance of any embodiment presented above, the one or more additional therapeutic agents are antifungal agents. In furtherance of any embodiment presented above, the one or more antifungal agents is selected from itraconazole, posaconazole, ketoconazole, clotrimazole, miconazole, voriconazole, caspofungin, anidulafungin, micafungin, nystatin, amphotericin b, griseofulvin, terbinafine, and/or flucytosine. In furtherance of any embodiment presented above, the disclosure provides a method of treating a subject suspected of having or at risk of developing a microbial infection comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a stimuli-responsive polysaccharide

antimicrobial agent described above. In furtherance of any embodiment presented above, the subject is suspected of having or at risk of developing a bacterial and/or a fungal infection. In furtherance of any embodiment presented above, the bacterial infection is caused by a bacterium selected from the group consisting of Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis,

Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enter ocolitica, and Yersinia pseudotuberculosis. In furtherance of any embodiment presented above, the fungal infection is caused by a fungus selected from the group consisting oiAbsidia corymbifera, Absidia ramose, Achorion gallinae, Actinomadura spp., Ajellomyces dermatididis , Aleurisma brasiliensis, Allersheria boydii, Arthroderma spp., Aspergillus flavus, Aspergillus fumigatu, Basidiobolus spp, Blastomyces spp, Cadophora spp, Candida albicans, Cercospora apii, Chrysosporium spp, Cladosporium spp, Cladothrix asteroids, Coccidioides immitis, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Cunninghamella elegans, Dematium wernecke, Discomyces israelii, Emmonsia spp, Emmonsiella capsulate, Endomyces geotrichum, Entomophthora coronate, Epidermophyton floccosum, Filobasidiella neoformans, Fonsecaea spp., Geotrichum candidum, Glenospora khartoumensis, Gymnoascus gypseus,

Haplosporangium parvum, Histoplasma, Histoplasma capsulatum, Hormiscium dermatididis, Hormodendrum spp., Keratinomyces spp, Longer onia soudanense, Leptosphaeria senegalensis, Lichtheimia corymbifera, Lobmyces loboi., Loboa loboi, Lobomycosis, Madurella spp., Malassezia furfur, Micrococcus pelletieri, Microsporum spp, Monilia spp., Mucor spp., Mycobacterium tuberculosis, Nannizzia spp., Neotestudina rosatii, Nocardia spp., Oidium albicans, Oospora lactis, Paracoccidioides brasiliensis, Petriellidium boydii, Phialophora spp., Piedraia hortae, Pityrosporum furfur, Pneumocystis jirovecii (or

Pneumocystis carinii), Pullularia gougerotii, Pyrenochaeta romeroi, Rhinosporidium seeberi, Sabouraudites (Microsporum) , Sartorya fumigate, Sepedonium, Sporotrichum spp., Stachybotrys, Stachybotrys chartarum, Streptomyce spp., Tinea spp., Torula spp,

Trichophyton spp, Trichosporon spp, and Zopfla rosatii. In furtherance of any embodiment presented above, the disclosure provides a method of treating a subject suspected of having or at risk of developing a microbial infection comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a stimuli-responsive polysaccharide antimicrobial agent described above and concurrently or sequentially, administering one or more antibiotics, and/or antifungal agents. In furtherance of any embodiment presented above, the one or more antibiotics is selected from Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Streptomycin, Spectinomycin, Geldanamycin, Herbimycin, Rifaximin, Loracarbef, Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin, Cephradine, Cephapirin,

Cephalothin, Cefalexin, Cefaclor, Cefoxitin, Cefotetan, Cefamandole, Cefmetazole,

Cefonicid, Loracarbef, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Moxalactam, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Dalbavancin, Oritavancin, Clindamycin, Lincomycin, Daptomycin, Azithromycin,

Clarithromycin, Roxithromycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Azlocillin,

Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin, Ticarcillin, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, Ticarcillin/clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin,

Lomefioxacin, Moxifloxacin, Nadifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,

Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole,

Sulfonamidochrysoidine, Demeclocycline, Doxycycline, Metacycline, Minocycline,

Oxy tetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine,

Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline,

Tinidazole, and Trimethoprim. In furtherance of any embodiment presented above, the one or more antifungal agents is selected from itraconazole, posaconazole, ketoconazole, clotrimazole, miconazole, voriconazole, caspofungin, anidulafungin, micafungin, nystatin, amphotericin b, griseofulvin, terbinafine, and/or flucytosine. In furtherance of any embodiment presented above, the disclosure provides a method of treating a subject suspected of having or at risk of developing a microbial infection comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a composition described above. In furtherance of any embodiment presented above, the subject is suspected of having or at risk of developing a bacterial and/or a fungal infection. In furtherance of any embodiment presented above, the bacterial infection is caused by a bacterium selected from the group consisting of Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcer ans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis,

Haplosporangium parvum, Histoplasma, Histoplasma capsulatum, Hormiscium dermatididis , Hormodendrum spp., Keratinomyces spp, Longer onia soudanense, Leptosphaeria senegalensis, Lichtheimia corymbifera, Lobmyces loboi., Loboa loboi, Lobomycosis, Madurella spp., Malassezia furfur, Micrococcus pelletieri, Microsporum spp, Monilia spp., Mucor spp., Mycobacterium tuberculosis, Nannizzia spp., Neotestudina rosatii, Nocardia spp., Oidium albicans, Oospora lactis, Paracoccidioides brasiliensis, Petriellidium boydii, Phialophora spp., Piedraia hortae, Pityrosporum furfur, Pneumocystis jirovecii (or

Pneumocystis carinii), Pullularia gougerotii, Pyrenochaeta romeroi, Rhinosporidium seeberi, Sabouraudites (Microsporum), Sartorya fumigate, Sepedonium, Sporotrichum spp., Stachybotrys, Stachybotrys chartarum, Streptomyce spp., Tinea spp., Torula spp,

Trichophyton spp, Trichosporon spp, and Zopfla rosatii. In furtherance of any embodiment presented above, the subject is suspected of having an intracellular bacterial infection. In furtherance of any embodiment presented above, the intracellular bacterial infection is caused by bacterium selected from the genus Chlamydophila, Ehrlichia, Rickettsia, Chlamydia, Salmonella, Neisseria, Brucella, Mycobacterium, Nocardia, Listeria, Francisella, Legionella, or Yersinia pestis. In furtherance of any embodiment presented above, the disclosure also provides a method of treating a subject suspected of having or at risk of developing a microbial infection comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a pharmaceutical composition described above. In furtherance of any embodiment presented above, the subject is suspected of having or at risk of developing a bacterial and/or a fungal infection. In furtherance of any embodiment presented above, the bacterial infection is caused by a bacterium selected from the group consisting of Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium,

Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enter ocolitica, and Yersinia pseudotuberculosis. In furtherance of any embodiment presented above, the fungal infection is caused by a fungus selected from the group consisting of Absidia corymbifera, Absidia ramose, Achorion gallinae, Actinomadura spp., Ajellomyces dermatididis, Aleurisma brasiliensis, Allersheria boydii, Arthroderma spp., Aspergillus flavus, Aspergillus fumigatu, Basidiobolus spp, Blastomyces spp, Cadophora spp, Candida albicans, Cercospora apii, Chrysosporium spp, Cladosporium spp, Cladothrix asteroids, Coccidioides immitis, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Cunninghamella elegans, Dematium wernecke, Discomyces israelii, Emmonsia spp, Emmonsiella capsulate, Endomyces geotrichum, Entomophthora coronate, Epidermophyton floccosum, Filobasidiella neoformans, Fonsecaea spp., Geotrichum candidum, Glenospora khartoumensis ,

Gymnoascus gypseus, Haplosporangium parvum, Histoplasma, Histoplasma capsulatum, Hormiscium dermatididis, Hormodendrum spp., Keratinomyces spp, Langeronia soudanense, Leptosphaeria senegalensis, Lichtheimia corymbifera, Lobmyces loboi., Loboa loboi, Lobomycosis, Madurella spp., Malassezia furfur, Micrococcus pelletieri, Microsporum spp, Monilia spp., Mucor spp., Mycobacterium tuberculosis, Nannizzia spp., Neotestudina rosatii, Nocardia spp., Oidium albicans, Oospora lactis, Paracoccidioides brasiliensis, Petriellidium boydii, Phialophora spp., Piedraia hortae, Pityrosporum furfur, Pneumocystis jirovecii (or Pneumocystis carinii), Pullularia gougerotii, Pyrenochaeta romeroi, Rhinosporidium seeberi, Sabouraudites (Microsporum), Sartorya fumigate, Sepedonium, Sporotrichum spp., Stachybotrys, Stachybotrys chartarum, Streptomyce spp., Tinea spp., Torula spp,

Trichophyton spp, Trichosporon spp, and Zopfla rosatii. In furtherance of any embodiment presented above, the disclosure also provides a method to prevent an infection by a microbial agent of a wound or a bum, comprising, consisting essentially of, or consisting of topically administering to the subject a therapeutically effective amount of a stimuli-responsive polysaccharide antimicrobial agent described above. In furtherance of any embodiment presented above, the disclosure also provides a method to prevent an infection by a microbial agent of a wound or a bum, comprising topically administering to the subject a

therapeutically effective amount of a composition described above. In furtherance of any embodiment presented above, the disclosure also provides a method to prevent an infection by a microbial agent of a wound or a bum, comprising, consisting essentially of, or consisting of topically administering to the subject a therapeutically effective amount of a

pharmaceutical composition described above. In furtherance of any embodiment presented above, disclosure also provides a method for inhibiting the growth of a microorganism or microbe by contacting the microorganism or microbe with an inhibiting effective amount of a stimuli-responsive polysaccharide antimicrobial agent described above. In furtherance of any embodiment presented above, the microorganism or microbe is contacted in vitro with the stimuli-responsive polysaccharide antimicrobial agent. In furtherance of any embodiment presented above, the microorganism or microbe is contacted in vivo with the stimuli- responsive polysaccharide antimicrobial agent.

[ 0007 ] In furtherance of any embodiment presented above or throughout the disclosure, the disclosure contemplates the use of a stimuli-responsive polysaccharide antimicrobial agent disclosed herein or any composition, including pharmaceutical compositions, which comprise, consist essentially of, or consist of a stimuli-responsive polysaccharide antimicrobial agent disclosed herein for use in preparing a medicament for the treatment of a microbial infection, or any disease or disorder resulting therefrom.

DESCRIPTION OF DRAWINGS

[ 0008 ] Figure 1 provides a schematic illustration of the current drug discovery paradigm in antibiotics. A target microbe that requires new antibiotics becomes the main subject of research. Typically, the new antibiotics are usually discovered in soil or marine bacteria, and synthetic derivatives are generated therefrom. While at first effective, the new antibiotics only have up to 14 years of efficacy before resistance is developed based on current trends.

[ 0009] Figure 2 provides a schematic illustration of the mechanisms of action for nanoantibiotics (nAbts) in comparison to mechanisms of action for conventional antibiotics for disrupting pathogenic organisms. Typically, mechanisms of action for nAbts involve cell membrane disruption; oxidation of cellular components caused by reactive oxygen species (ROS); interruption of transmembrane electron transport; and inducing mitochondria and DNA damage via heavy metal ions and ROS. In contrast, mechanisms of action for conventional antibiotics involve inhibiting nucleic acid transcription and function; hindering protein synthesis; disrupting general cell synthesis or function; disrupting selective membrane permeability; or interfering with the synthesis of key biological components such as folic acid.

[ 0010 ] Figure 3 presents a schematic illustration of the mode of action against gram

(+) bacteria for chitosan and oligo-chitosan.

[ 0011 ] Figure 4 presents a schematic illustration of the mode of action against gram

(-) bacteria for chitosan and oligo-chitosan.

[ 0012 ] Figure 5 presents a schematic illustration of the mode of action against fungi for chitosan and oligo-chitosan

[ 0013] Figure 6 indicates the minimum inhibitory concentration of native chitosan against gram (+) bacteria

[ 0014 ] Figure 7 indicates the minimum inhibitory concentration of native chitosan against gram (-) bacteria.

[ 0015] Figure 8 indicates the minimum inhibitory concentration of native chitosan against different fungi.

[ 0016] Figure 9 provides an embodiment of the synthesis scheme for an acid- transforming chitosan (ATC).

[ 0017 ] Figure lOA-C provides for the molecular characterizations of chitosan, Phth-

TFA-C, and ATC by 1H NMR (A), FTIR (B), and MALDI-TOF (C).

[ 0018 ] Figure 11A-B shows the acid-triggered transformation of ATC to chitosan

(marked as C) at an acidic pH (10 mg/mL), confirmed by (A) 1H NMR and (B) solubility changes upon acid-transformation and subsequent neutralization, as also quantified by the percentage of solubilized polymer (10 mg added in 1 mL of DI water and buffers).

[ 0019] Figure 12 presents inverted microscope images of chitosan and ATC after incubation at pH 5.0 and neutralization as described in FIG. 11B.

[ 0020 ] Figure 13 provides for the hydrolysis kinetics of free ATC and ATC/siRNA polyplexes at pH 5.0, 6.0, and 7.4. Half-lives of ATC and ATC/siRNA polyplexes at different pHs were calculated using the Arrhenius equation where A and represented the integrations of ketal linkage peaks (1.46 ppm) in ¾ NMR spectrum. ATC (10 mg) and ATC/siRNA (N/P 50; 0.85mg ATC and 15 μg siRNA) were dissolved in 1 mL of acetate (pH 5.0 and pH 6.0, adjusted by NaOH) and Tris-DCL (pH 7.4) buffer in D2O, respectively, and incubated at 37 °C for various periods of time. Note that y-axis scales for the two charts are significantly different.

[ 0021 ] Figure 14A-D shows ATC/siRNA polyplexes prepared at different N/P ratios and their characterization by (A) DLS size measurement and zeta-potential analysis, (B) ethidium bromide (EtBr) exclusion assay, (C) gel retardation assay before and after acid- hydrolysis of polyplexes (S: free siRNA standard), and (D) TEM.

[ 0022 ] Figure 15 presents fluorescence microscope images and flow cytometry data of eGFP-expressing HeLa cells after 72 h of incubation with 20 μΜ ATC/eGFP siRNA polyplexes prepared at N/P ratio of 50. The almost completely muted expression of eGFP in the cells indicated highly efficient transfection by ATC/eGFP siRNA polyplexes, significantly higher than that by PEI/eGFP siRNA polyplexes (N/P 10).

[ 0023] Figure 16 provides a schematic of environmental regulation of virulent factors matched with stimuli classification. Microbes' virulent factors can be regulated by environmental signals, which can be classified as chemical, biological, or physical.

[ 0024 ] Figure 17A-B provides a schematic of the formation of a stimuli-responsive nanoantibiotics particle: (A) combined system can be created by combining conventional antibiotics with nanoantibiotics. Stimuli response can aid in the synergistic efficacy by allowing a particle to display multiple therapeutic effects based on the stimulus applied. (B) Proposed therapeutic effects of stimuli-responsive nanoantibiotics (sr-nAbts) against intracellular infection. To target intracellular microbes, conventional antibiotics are combined with sr-nAbts. Upon endocytosis, environmental effects (e.g. , acidic pH) trigger the particle to unpack to escape the endosome, the released nonantibiotics and conventional antibiotics then target the microbe. Based on the infection, a second trigger can release another drug and use an additional nAbts release mechanism

[ 0025] Figure 18 demonstrates the dose dependent toxicity of acid-transforming chitosan (ATC) samples versus E. Coli.

[ 0026] Figure 19 provides time courses of the toxicity of ATC vs. chitosan in E. Coli.

[ 0027 ] Figure 20 provides the results of MTT assays looking at the toxicity of ATC vs. chitosan in mammalian cell lines, human Hela cells and mouse Raw 264 cells.

[ 0028 ] Figure 21 provides the results of in vivo assays in C57BL/6 mice looking at

ATC vs. chitosan toxicity over a period of 20 days. [ 0029] Figure 22A-C provides for the antimicrobial efficacy and toxicity of ATC.

(A) ATC polymer against S. typhimurium varying pH conditions. (B) MTT assay of Chitosan and ATC on RAW 264.7 cells. (C) Comparison of the in vivo toxicity of chitosan vs ATC.

[ 0030 ] Figure 23 looks at the formation of ATC/pdm polyplexes. ATC/pdrn polyplex characterizations at N/P = 100 were shown by DLS data and TEM images.

[ 0031 ] Figure 24 demonstrates the utilization of ATC/pdrn polyplexes to treat acute intracellular infections. RAW 264.7 cells infected for 1 h with GFP S. typhimurium and then treated with ATC/pdrn polyplexes at varying polymer concentrations. The results of which are reflected in the presented flow cytometry data and corresponding fluorescence images, (p O.05)

[ 0032 ] Figure 25 demonstrates the utilization of ATC/pdrn polyplexes to treat prolonged intracellular infections. RAW 264.7 cells infected for 16 h (prolonged) with GFP S. typhimurium then treated with ATC/pdrn polyplexes at varying polymer concentrations, flow cytometry data indicating the median fluorescence intensity (MFI) with corresponding fluorescence images. (p<0.05)

[ 0033] Figure 26A-B presents the colony count of S. typhimurium post treatment of

RAW 264.7 cells with ATC/pdrn polyplexes followed by cell lysis. (A) Agar plate of titrated colonies, with the (B) overall count of the colonies at each titration compared side-by-side.

[ 0034 ] Figure 27 provides for the cationic polymer antimicrobial efficacy of ATC,

PEI and OCMC at pH 5.5 and 7.

DETAILED DESCRIPTION

[ 0035] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibiotic" includes a plurality of such antibiotics and reference to "the polysaccharide" includes reference to one or more polysaccharides and equivalents thereof known to those skilled in the art, and so forth.

[ 0036] Also, the use of "or" means "and/or" unless stated otherwise. Similarly,

"comprise," "comprises," "comprising" "include," "includes," and "including" are interchangeable and not intended to be limiting.

[ 0037 ] It is to be further understood that where descriptions of various embodiments use the term "comprising," those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of or "consisting of." [ 0038 ] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although many methods and reagents are similar or equivalent to those described herein, the exemplary methods and materials are disclosed herein.

[ 0039] All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the methodologies, which might be used in connection with the description herein. Moreover, with respect to any term that is presented in one or more publications that is similar to, or identical with, a term that has been expressly defined in this disclosure, the definition of the term as expressly provided in this disclosure will control in all respects.

[ 0040 ] In regards to various lists provided herein, including lists of antibiotics, microorganisms, antifungals, etc. the disclosure fully contemplates in various aspects that select members may be selected from the foregoing lists, including, specific combinations of antibiotics, specific combinations of bacteria (e.g. , gram positive vs. gram negative bacteria), specific combinations of antifungal agents, specific combinations of fungi. As such, the disclosure fully contemplates the use of subsets of members selected from the various lists presented herein in various combinations.

[ 0041 ] The term "about" or "approximately," as used herein, in reference to a number are generally taken to include numbers that fall within a range of 5% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Where ranges are stated, the endpoints are included within the range unless otherwise stated or otherwise evident from the context.

[ 0042 ] The term "alkenyl", refers to an organic group that is comprised of carbon and hydrogen atoms that contains at least one double covalent bond between two carbons.

Typically, an "alkenyl" as used in this disclosure, refers to organic group that contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 carbon atoms, or any range of carbon atoms between or including any two of the foregoing values. While a C2-alkenyl can form a double bond to a carbon of a parent chain, an alkenyl group of three or more carbons can contain more than one double bond. It certain instances the alkenyl group will be conjugated, in other cases an alkenyl group will not be conjugated, and yet other cases the alkenyl group may have stretches of conjugation and stretches of nonconjugation.

Additionally, if there is more than 2 carbon, the carbons may be connected in a linear manner, or alternatively if there are more than 3 carbons then the carbons may also be linked in a branched fashion so that the parent chain contains one or more secondary, tertiary, or quaternary carbons. An alkenyl may be substituted or unsubstituted, unless stated otherwise. Examples of substitutions for alkenyls include, but are not limited, to halos, hydroxyls, anhydrides, carbonyls, carboxyls, carbonates, carboxylates, aldehydes, haloformyls, esters, hydroperoxy, peroxy, ethers, orthoesters, carboxamides, amines, imines, imides, azides, azos, cyanates, isocyanates, nitrates, nitriles, isonitriles, nitrosos, nitros, nitrosooxy, pyridyls, sulfhydryls, sulfides, disulfides, sulfinyls, sulfos, thiocyanates, isothiocyanates,

carbonothioyls, phosphinos, phosphonos, phosphates, and silyl ethers.

[ 0043] The term "alkyl", refers to an organic group that is comprised of carbon and hydrogen atoms that contains single covalent bonds between carbons. Typically, an "alkyl" as used in this disclosure, refers to an organic group that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 carbon atoms, or any range of carbon atoms between or including any two of the foregoing values. Where if there is more than 1 carbon, the carbons may be connected in a linear manner, or alternatively if there are more than 2 carbons then the carbons may also be linked in a branched fashion so that the parent chain contains one or more secondary, tertiary, or quatemary carbons. An alkyl may be substituted or unsubstituted, unless stated otherwise. Examples of substitutions for alkyls include, but are not limited, to halos, hydroxyls, anhydrides, carbonyls, carboxyls, carbonates, carboxylates, aldehydes, haloformyls, esters, hydroperoxy, peroxy, ethers, orthoesters, carboxamides, amines, imines, imides, azides, azos, cyanates, isocyanates, nitrates, nitriles, isonitriles, nitrosos, nitros, nitrosooxy, pyridyls, sulfhydryls, sulfides, disulfides, sulfinyls, sulfos, thiocyanates, isothiocyanates, carbonothioyls, phosphinos, phosphonos, phosphates, and silyl ethers.

[ 0044 ] The term "alkynyl", refers to an organic group that is comprised of carbon and hydrogen atoms that contains a triple covalent bond between two carbons. Typically, an "alkynyl" as used in this disclosure, refers to organic group that contains that contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 carbon atoms, or any range of carbon atoms between or including any two of the foregoing values. While a C2-alkynyl can form a triple bond to a carbon of a parent chain, an alkynyl group of three or more carbons can contain more than one triple bond. Where if there is more than 3 carbon, the carbons may be connected in a linear manner, or alternatively if there are more than 4 carbons then the carbons may also be linked in a branched fashion so that the parent chain contains one or more secondary, tertiary, or quaternary carbons. An alkynyl may be substituted or unsubstituted, unless stated otherwise. Examples of substitutions for alkynyls include, but are not limited, to halos, hydroxyls, anhydrides, carbonyls, carboxyls, carbonates, carboxylates, aldehydes, haloformyls, esters, hydroperoxy, peroxy, ethers, orthoesters, carboxamides, amines, imines, imides, azides, azos, cyanates, isocyanates, nitrates, nitriles, isonitriles, nitrosos, nitros, nitrosooxy, pyridyls, sulfhydryls, sulfides, disulfides, sulfinyls, sulfos, thiocyanates, isothiocyanates, carbonothioyls, phosphinos, phosphonos, phosphates, and silyl ethers.

[ 0045] The term "aryl", as used in this disclosure, refers to a conjugated planar ring system with delocalized pi electron clouds that contain only carbon as ring atoms. An "aryl" for the purposes of this disclosure encompass from 1 to 4 aryl rings wherein when the aryl is greater than 1 ring the aryl rings are joined so that they are linked, fused, or a combination thereof. An aryl may be substituted or unsubstituted, or in the case of more than one aryl ring, one or more rings may be unsubstituted, one or more rings may be substituted, or a combination thereof. Examples of substitutions for aryls include, but are not limited, to halos, hydroxyls, anhydrides, carbonyls, carboxyls, carbonates, carboxylates, aldehydes, haloformyls, esters, hydroperoxy, peroxy, ethers, orthoesters, carboxamides, amines, imines, imides, azides, azos, cyanates, isocyanates, nitrates, nitriles, isonitriles, nitrosos, nitros, nitrosooxy, pyridyls, sulfhydryls, sulfides, disulfides, sulfinyls, sulfos, thiocyanates, isothiocyanates, carbonothioyls, phosphinos, phosphonos, phosphates, and silyl ethers.

[ 0046] The term "CRISPR-Cas system", "CRISPRi system" or "CRISPR-Cpfl system" as used herein refers to all the components that can be or are used to perform gene editing using a CRISPR gene editing system, including components, such as plasmids, guide RNAs (e.g. , crRNAs), Cas proteins (e.g. , Cas-3, Cas-9), and Cpfl proteins.

[ 0047 ] The term "cylcloalkyl", as used in this disclosure, refers to an alkyl that contains at least 3 carbon atoms but no more than 12 carbon atoms connected so that it forms a ring. A "cycloalkyl" for the purposes of this disclosure encompasses from 1 to 4 cycloalkyl rings, wherein when the cycloalkyl is greater than 1 ring, then the cycloalkyl rings are joined so that they are linked, fused, or a combination thereof. A cycloalkyl may be substituted or unsubstituted, or in the case of more than one cycloalkyl ring, one or more rings may be unsubstituted, one or more rings may be substituted, or a combination thereof. Examples of substitutions for cycloalkyls include, but are not limited, to halos, hydroxyls, anhydrides, carbonyls, carboxyls, carbonates, carboxylates, aldehydes, haloformyls, esters, hydroperoxy, peroxy, ethers, orthoesters, carboxamides, amines, imines, imides, azides, azos, cyanates, isocyanates, nitrates, nitriles, isonitriles, nitrosos, nitros, nitrosooxy, pyridyls, sulfhydryls, sulfides, disulfides, sulfinyls, sulfos, thiocyanates, isothiocyanates, carbonothioyls, phosphinos, phosphonos, phosphates, and silyl ethers.

[ 0048 ] The term "(C_x-C_y)" where x and y are integers and y > x, refers to a functional group which comprises a range of carbon atoms specified by x and y. For example, a "(Ci- Ci2)alkyl" refers to an alkyl group which has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. For purposes of this disclosure, a range specified by "(C_x-C_y)" includes inclusive ranges encompassed therein, unless specifically specified otherwise. For example, "(Ci- C12)" includes ranges such as (Ci-C₆), (C2-C12), (C3-C11), (C4-C10), etc.

[ 0049] The term "hetero-" when used as a prefix, such as, hetero-alkyl, hetero- alkenyl, hetero-alkynyl, or hetero-hydrocarbon, for the purpose of this disclosure refers to the specified hydrocarbon having one or more carbon atoms replaced by non-carbon atoms as part of the parent chain. Examples of such non-carbon atoms include, but are not limited to, N, O, S, Si, Al, B, and P. If there is more than one non-carbon atom in the hetero-based parent chain then this atom may be the same element or may be a combination of different elements, such as N and O. In furtherance of any embodiment presented herein, a "hetero"- hydrocarbon (e.g. , alkyl, alkenyl, alkynyl) refers to a hydrocarbon that has from 1 to 3 C, N and/or S atoms as part of the parent chain.

[ 0050 ] The term "disorder" as used herein is intended to be generally synonymous, and is used interchangeably with, the terms "disease," "syndrome," and "condition" (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms.

[ 0051 ] The term "heterocycle," as used herein, refers to ring structures that contain at least 1 noncarbon ring atom. A "heterocycle" for the purposes of this disclosure encompass from 1 to 4 heterocycle rings, wherein when the heterocycle is greater than 1 ring the heterocycle rings are joined so that they are linked, fused, or a combination thereof. A heterocycle may be aromatic or nonaromatic, or in the case of more than one heterocycle ring, one or more rings may be nonaromatic, one or more rings may be aromatic, or a combination thereof. A heterocycle may be substituted or unsubstituted, or in the case of more than one heterocycle ring one or more rings may be unsubstituted, one or more rings may be substituted, or a combination thereof. Typically, the noncarbon ring atom is N, O, S, Si, Al, B, or P. In the case where there is more than one noncarbon ring atom, these noncarbon ring atoms can either be the same element, or combination of different elements, such as N and O. Examples of heterocycles include, but are not limited to: a monocyclic heterocycle such as, aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazolidine, pyrazolidine, pyrazoline, dioxolane, sulfolane 2,3-dihydrofuran, 2,5-dihydrofuran tetrahydrofuran, thiophane, piperidine, 1, 2,3, 6-tetrahydro-pyri dine, piperazine, morpholine, thiomorpholine, pyran, thiopyran, 2,3-dihydropyran,

tetrahydropyran, 1,4-dihydropyridine, 1,4-dioxane, 1,3-dioxane, dioxane, homopiperidine, 2,3,4,7-tetrahydro-lH-azepine homopiperazine, 1,3-dioxepane, 4,7-dihydro-l,3-dioxepin, and hexamethylene oxide; and poly cyclic heterocycles such as, indole, indoline, isoindoline, quinoline, tetrahydroquinoline, isoquinoline, tetrahydroisoquinoline, 1,4-benzodioxan, coumarin, dihydrocoumarin, benzofuran, 2,3-dihydrobenzofuran, isobenzofuran, chromene, chroman, isochroman, xanthene, phenoxathiin, thianthrene, indolizine, isoindole, indazole, purine, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, phenanthridine, perimidine, phenanthroline, phenazine, phenothiazine, phenoxazine, 1 ,2- benzisoxazole, benzothiophene, benzoxazole, benzthiazole, benzimidazole, benztriazole, thioxanthine, carbazole, carboline, acridine, pyrolizidine, and quinolizidine. In addition to the poly cyclic heterocycles described above, heterocycle includes poly cyclic heterocycles wherein the ring fusion between two or more rings includes more than one bond common to both rings and more than two atoms common to both rings. Examples of such bridged heterocycles include quinuclidine, diazabicyclo[2.2.1]heptane and 7- oxabicyclo[2.2.1]heptane.

[ 0052 ] The terms "heterocyclic group", "heterocyclic moiety", "heterocyclic", or

"heterocyclo" used alone or as a suffix or prefix, refers to a heterocycle that has had one or more hydrogens removed therefrom.

[ 0053] The term "hydrocarbons" refers to groups of atoms that contain only carbon and hydrogen. Examples of hydrocarbons that can be used in this disclosure include, but are not limited to, alkanes, alkenes, alkynes, arenes, and benzyls.

[ 0054 ] The term "non-release controlling excipient" as used herein, refers to an excipient whose primary function do not include modifying the duration or place of release of the active substance from a dosage form as compared with a conventional immediate release dosage form. [ 0055] The term "microorganism" or "microbe" refers to a microscopic organism, especially a bacterium, virus, or fungus.

[ 0056] The term "optionally substituted" refers to a functional group, typically a hydrocarbon or heterocycle, where one or more hydrogen atoms may be replaced with a substituent. Accordingly, "optionally substituted" refers to a functional group that is substituted, in that one or more hydrogen atoms are replaced with a substituent, or unsubstituted, in that the hydrogen atoms are not replaced with a substituent. For example, an optionally substituted hydrocarbon group refers to an unsubstituted hydrocarbon group or a substituted hydrocarbon group.

[ 0057 ] The term "optionally substituted" with respect to hydrocarbons, heterocycles, and the like, refers to structures that may be substituted, or alternatively be unsubstituted. Examples of optional substitutions include, but are not limited, to halos, hydroxyls, anhydrides, carbonyls, carboxyls, carbonates, carboxylates, aldehydes, haloformyls, esters, hydroperoxy, peroxy, ethers, orthoesters, carboxamides, amines, imines, imides, azides, azos, cyanates, isocyanates, nitrates, nitriles, isonitriles, nitrosos, nitros, nitrosooxy, pyridyls, sulfhydryls, sulfides, disulfides, sulfinyls, sulfos, thiocyanates, isothiocyanates,

carbonothioyls, phosphinos, phosphonos, phosphates, and silyl ethers.

[ 0058 ] The term "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" as used herein, refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each component must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. Examples of "pharmaceutically acceptable carriers" and "pharmaceutically acceptable excipients" can be found in the following, Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins:

Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Rowe et al , Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, Fla., 2004. [ 0059] The terms "polynucleotide" and "oligonucleotide" as used herein refer to the meaning as is generally accepted in the art. The terms generally refer to a chain of nucleotides. "Nucleic acids" and "nucleic acid molecules" are polymers of nucleotides. Thus, "nucleic acids," "polynucleotides" and "oligonucleotides" are interchangeable herein. One skilled in the art has the general knowledge that nucleic acids are polynucleotides which can be hydrolyzed into monomeric nucleotides. Monomeric nucleotides can be further hydrolyzed into nucleosides.

[ 0060 ] The term "release controlling excipient" as used herein, refers to an excipient whose primary function is to modify the duration or place of release of the active substance from a dosage form as compared with a conventional immediate release dosage form.

[ 0061 ] The term "RNA" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a molecule comprising at least one ribofuranoside residue, such as a ribonucleotide. The term "ribonucleotide" means a nucleotide with a hydroxyl group at the 2' position of a β-D-ribofuranose moiety. The term refers to a double- stranded RNA, a single-stranded RNA, an isolated RNA such as a partially purified RNA, an essentially pure RNA, a synthetic RNA, a recombinantly -produced RNA, or an altered RNA that differs from a naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides therein. Such alterations can include addition of non- nucleotide material, for example, at one or more non-terminal nucleotides of an RNA molecule. As such, nucleotides in the single-stranded RNA molecules of the invention can comprise non-standard nucleotides, such as non-naturally occurring nucleotides, chemically- synthesized and/or modified nucleotides, or deoxynucleotides.

[ 0062 ] The term "therapeutically acceptable" refers to those compounds (or salts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, immunogenicity, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.

[ 0063] The terms "treat", "treating" and "treatment", as used herein, refers to ameliorating symptoms associated with a disease or disorder (e.g. , multiple sclerosis), including preventing or delaying the onset of the disease or disorder symptoms, and/or lessening the severity or frequency of symptoms of the disease or disorder.

[ 0064 ] The term "subject" as used herein, refers to an animal, including, but not limited to, a primate (e.g. , human, monkey, chimpanzee, gorilla, and the like), rodents (e.g. , rats, mice, gerbils, hamsters, ferrets, and the like), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, and the like. The terms "subject" and "patient" are used interchangeably herein. For example, a mammalian subject can refer to a human patient.

[ 0065] The term "substituent" refers to an atom or group of atoms substituted in place of a hydrogen atom. For purposes of this invention, a substituent would include deuterium atoms.

[ 0066] The term "substituted" with respect to hydrocarbons, heterocycles, and the like, refers to structures wherein the parent chain contains one or more substituents.

[ 0067 ] The term "unsubstituted" with respect to hydrocarbons, heterocycles, and the like, refers to structures wherein the parent chain contains no substituents.

[ 0068 ] As used herein, a wavy line intersecting another line that is connected to an atom indicates that this atom is covalently bonded to another entity that is present but not being depicted in the structure. A wavy line that does not intersect a line but is connected to an atom indicates that this atom is interacting with another atom by a bond or some other type of identifiable association.

[ 0069] It is clear that there is a desperate need for innovative therapeutics against drug resistant microbes. While the new antimicrobials are effective and promising, they generally provide only a short-term solution to the overall challenge of drug-resistant microbes. It takes anywhere from of a few months to approximately ten years for a resistant gene to develop. Aside from environmental pressure and overuse, another cause for this rapid loss of efficacy is because most available antibiotics are derived from a compound discovered in a microbe for an example, penicillin (e.g. , see FIG. 1). While the source microbe is resistant against the compound, the unfortunate reality is that the resistant gene can be easily passed on through horizontal gene transfer to a target microbe. To truly gain an advantage against microbes, antimicrobial agents have to be engineered and designed to avoid that cycle.

[ 0070 ] Recently, the use of nanoantibiotics as a therapeutic strategy has gained a lot of attention. Nanoantibiotics (nAbts) are nanomaterials that have an antimicrobial activity or improve the efficacy and safety of administering antibiotics. nAbts possess many advantages over conventional antibiotics, including but not limited to production, storage, durability, and versatility. Nanoantibiotic polymers typically need to be formulated into nanoparticles for full usage of the therapeutic antimicrobial properties. These nanoantibiotic polymers hinder the growth of bacteria through one of the nAbts mechanisms (e.g. , see FIG. 2). Chitosan, a nonantibiotic polymer, is a partially deacetylated chitin that has been shown to possess a wide spectrum of antimicrobial activity. Chitosan's antimicrobial mechanisms include: increasing the permeability of the microbial cell wall, and the chelation of trace metals. Chitosan has demonstrated efficacy against fungi (e.g. , see FIGs. 5 and 8), gram-positive bacteria (e.g. , see FIGs. 3 and 6), and gram-negative bacteria (e.g. , see FIGs. 4 and 7). According, chitosan is recognized as a broad spectrum antimicrobial agent.

[ 0071 ] Although chitosan possesses excellent antimicrobial properties, there are some challenges involved in its use. For one, chitosan is soluble in an acidic environment, but is not soluble in the biologically relevant pH range of 7-8. Accordingly, the lack of solubility limits the general use of chitosan in physiological settings, e.g. , use in the blood stream.

[ 0072 ] In order to avoid the foregoing drawbacks, the stimuli-responsive

polysaccharide antimicrobial agents of the disclosure comprise a chitosan-based backbone in which a pH responsive linker has been appended to the chitosan backbone. pH responsive linkers exhibit unique advantages against microbes that thrive in highly acidic environments, whether extracellularly in the stomach (pH = 1-3), the gastrointestinal tract (pH = 5-8), or intracellularly in phagolysosomes (pH = 4.5-5) and macrophages. Moreover, the stimuli- responsive polysaccharide antimicrobial agents of the disclosure are well suited to treat chronic infections and wounds, which typically have pH values between 5.4 and 7.4.

[ 0073] While soluble formulations comprising chitosan may be possible, these formulations lack the advantages of the stimuli-responsive polysaccharide antimicrobial agents disclosed herein, including reversible but stable modification of the chitosan structure, and the maintenance of the native structure of chitosan. The reversibility of the stimuli- responsive polysaccharide antimicrobial agents disclosed herein provides additional advantages in fields where the antimicrobial characteristics are desired. For example, the stimuli-responsive polysaccharide antimicrobial agents of the disclosure are stable long enough to realize solubility benefits, while still exhibiting chitosan like antimicrobial properties. Further, as shown herein, the stimuli-responsive polysaccharide antimicrobial agents disclose herein exhibit low toxicity and maintain effective antimicrobial activity under physiological conditions. The stimuli-responsive polysaccharide antimicrobial agents of the disclosure can be used 'as is' in applications where direct application of polymeric antibiotic is desired, and/or where use of chitosan may be toxic at high doses.

[ 0074 ] In furtherance of any embodiment presented herein, the disclosure provides for a stimuli-responsive polysaccharide antimicrobial agent that comprises the core native structure of chitosan which has been modified with acid labile groups that can be cleaved in an acidic environment (i.e. , solutions that have a pH < 7.0). For purposes of this disclosure, "acid labile group" or "acid cleavable group" as used herein refers to a moiety which comprises a moiety that is designed to undergo hydrolysis in an acidic environment (pH < 7.0), such as the acidic environment of an endosome of phagosome. For example, ketal linkages rapidly cleave at a mildly acidic endosomal pH (~5.0) and have been intensively employed in the cytoplasmic release of biomacromolecules such as proteins and nucleic acids. Other exemplary acid labile groups comprise pH responsive groups, including but not limited to, acetal groups, ester groups, hydrazine groups, carboxy dimethyl maleic anhydride groups, orthoester groups, imine groups, β-thiopropionate groups, cis-aconityl groups, vinyl ether groups, and phosphoramidite groups. In furtherance of any embodiment presented herein, the disclosure provides for a stimuli-responsive polysaccharide antimicrobial agent that comprises a cationic, water soluble branch that has been grafted onto the primary hydroxyl groups of chitosan via an acid-labile linkage (e.g., a ketal linkage) resulting in acid- transforming chitosan (ATC). The chemical modification is designed to improve aqueous solubility of chitosan and to improve the efficacy of chitosan. The engineered stimuli- responsive polysaccharide antimicrobial agents of the disclosure exhibit good antimicrobial properties but are far more soluble than chitosan in the biologically relevant pH range of 7-8. Thus, the stimuli-responsive polysaccharide antimicrobial agents of the disclosure are a more effective nanoantibiotic polymer than chitosan itself.

[ 0075] In furtherance of any embodiment presented herein, the disclosure provides for a stimuli-responsive polysaccharide antimicrobial agent that comprises, consists essentially of, or consists of the structure of Formula I:

Formula I

or a pharmaceutically acceptable salt thereof,

wherein, AR¹ is an acid labile group, preferably selected from

and

AR² is an acid labile group, preferably selected from

R^R¹⁰ are each independently selected from H, -CH3, -CH2CH3, -OCH3, and - OCH2CH3, an optionally substituted (Ci-Ci2)alkyl group, an optionally substituted (Ci- Ci2)heteroalkyl group, an optionally substituted (Ci-Ci2)alkenyl group, an optionally substituted (Ci-Ci2)heteroalkenyl group, an optionally substituted (Ci-Ci2)alkynyl group, an optionally substituted (Ci-Ci2)heteroalkynyl group, an optionally substituted (C4- C8)cylcoalkyl group, an optionally substituted aryl group, or an optionally substituted heterocycle, wherein R² and R³ may be connected to each other to form a ring structure, preferably a cylcoalkyl or a heterocycle, and wherein R⁷ and R⁸ may be connected to form a ring structure, preferably a cylcoalkyl or a heterocycle,

n is an integer that is or is greater than 10, 50, 100, 200, 500, 800, 1000, 5000, 10000, or a range that includes or is between any two of the foregoing integers.

[ 0076] In furtherance of any embodiment presented herein, the disclosure provides for a stimuli-responsive polysaccharide antimicrobial agent that comprises, consists essentially of, or consists of the structure of Formula 1(a):

Formula 1(a) or a pharmaceutically acceptable salt thereof,

wherein,

R¹ is a substituted (Ci-Ci2)alkyl group, a substituted (Ci-Ci2)heteroalkyl group, a substituted (Ci-Ci2)alkenyl group, a substituted (Ci-Ci2)heteroalkenyl group, a substituted (Ci-Ci2)alkynyl group, a substituted (Ci-Ci2)heteroalkynyl group, a substituted (C₄- C8)cylcoalkyl group, a substituted aryl group, or an optionally substituted heterocycle, wherein R¹ comprises a substitution or group that can be protonated in an acidic environment;

R⁶ is a substituted (Ci-Ci2)alkyl group, a substituted (Ci-Ci2)heteroalkyl group, a substituted (Ci-Ci2)alkenyl group, a substituted (Ci-Ci2)heteroalkenyl group, a substituted (Ci-Ci2)alkynyl group, a substituted (Ci-Ci2)heteroalkynyl group, a substituted (C₄- C8)cylcoalkyl group, a substituted aryl group, or an optionally substituted heterocycle, wherein R⁶ comprises a substitution or group that can be protonated in an acidic environment;

R², R³, R⁷, and R⁸ are each independently selected from H, -CH₃, -CH2CH3, -OCH3,

2 and R³ may be connected together to form a ring structure selected

[ 0077 ] In furtherance of any embodiment presented herein, the disclosure provides for a stimuli-responsive polysaccharide antimicrobial agent that comprises, consists essentially of, or consists of the structure of Formula 1(b):

Formula 1(b)

or a pharmaceutically acceptable salt thereof,

wherein,

2 and R³ may be connected together to form a ring structure selected

and wherein R⁷ and R⁸ may be connected together to form a ring

structure selected from ^"

[ 0078 ] In furtherance of any embodiment presented herein, the disclosure provides for a stimuli-responsive polysaccharide antimicrobial agent that comprises, consists essentially of, or consists of the structure of Formula 1(c):

Formula 1(c)

or a pharmaceutically acceptable salt thereof,

wherein n is an integer that is or is greater than 10, 50, 100, 200, 500, 800, 1000, 5000, 10000, or a range that includes or is between any two of the foregoing integers.

[ 0079] Pharmaceutically acceptable salts comprise pharmaceutically-acceptable anions and/or cations. Pharmaceutically-acceptable cations include among others, alkali metal cations (e.g. , Li⁺, Na⁺, K⁺), alkaline earth metal cations (e.g., Ca²⁺, Mg²⁺), non-toxic heavy metal cations and ammonium (NH4⁺) and substituted ammonium (N(R')4⁺, where R is hydrogen, alkyl, or substituted alkyl, i.e. , including, methyl, ethyl, or hydroxy ethyl, specifically, trimethyl ammonium, triethyl ammonium, and triethanol ammonium cations). Pharmaceutically-acceptable anions include among other halides (e.g. , CI^", Br^"), sulfate, acetates (e.g., acetate, trifluoroacetate), ascorbates, aspartates, benzoates, citrates, and lactate.

[ 0080 ] The disclosure also provides a method for inhibiting the growth of a microorganism or microbe by contacting the microorganism or microbe with an inhibiting effective amount of a stimuli-responsive polysaccharide antimicrobial agent of the disclosure. The term "contacting" refers to exposing the microbe (e.g. , bacterium) to an antimicrobial agent so that the antimicrobial agent can inhibit, kill, or lyse the microbe or render it susceptible to oxidative destruction. Contacting of a microbe with a stimuli-responsive polysaccharide antimicrobial agent disclosed herein can occur in vitro, for example, by adding the agent to a bacterial culture, or contacting a microbial contaminated surface with an antimicrobial agent of the disclosure. Alternatively, contacting can occur in vivo, for example, by administering a stimuli-responsive polysaccharide antimicrobial agent disclosed herein to a subject (e.g. , a mammalian subject) suspected of having a microbial infection or susceptible to infection by a microbe (e.g. , a wound or burn). In vivo contacting of a microbe with a stimuli-responsive polysaccharide antimicrobial agent disclosed herein can be extracellular contacting the microbe and/or intracellular contacting the microbe depending on the pathogenesis of the microbe. "Inhibiting" or "inhibiting effective amount" refers to the amount of a stimuli-responsive polysaccharide antimicrobial agent that is sufficient to cause, for example, a bacteriostatic or bactericidal effect, or an antifungal effect. Bacteria that can be affected by the use of a stimuli-responsive polysaccharide antimicrobial agent disclosed herein include both gram-negative and gram-positive bacteria (see FIGs. 3, 4, 6 to 8). For example, bacteria that can be affected by the stimuli-responsive polysaccharide antimicrobial agent of the disclosure include, but are not limited to, Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus , Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enter ocolitica, and Yersinia pseudotuberculosis. Infection with one or more of these bacteria can result in diseases such as bacteremia, pneumonia, meningitis, osteomyelitis, endocarditis, sinusitis, arthritis, urinary tract infections, tetanus, gangrene, colitis, acute gastroenteritis, impetigo, acne, acne rosacea, wound infections, burn infections, fasciitis, bronchitis, and a variety of abscesses, nosocomial infections, and opportunistic infections. The method for inhibiting the growth of bacteria can also include contacting the bacterium with a stimuli-responsive polysaccharide antimicrobial agent disclosed herein in combination with one or more additional therapeutic agents, e.g. , antibiotics, gene silencing molecules, etc.

[ 0081 ] Fungal organisms may also be treated by a stimuli-responsive polysaccharide antimicrobial agent of the disclosure. Example of such fungal organisms include, but are not limited to, Absidia corymbifera, Absidia ramose, Achorion gallinae, Actinomadura spp., Ajellomyces dermatididis, Aleurisma brasiliensis, Allersheria boydii, Arthroderma spp., Aspergillus flavus, Aspergillus fumigatu, Basidiobolus spp, Blastomyces spp, Cadophora spp, Candida albicans, Cercospora apii, Chrysosporium spp, Cladosporium spp, Cladothrix asteroids, Coccidioides immitis, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Cunninghamella elegans, Dematium wernecke, Discomyces israelii, Emmonsia spp, Emmonsiella capsulate, Endomyces geotrichum, Entomophthora coronate, Epidermophyton floccosum, Filobasidiella neoformans, Fonsecaea spp., Geotrichum candidum, Glenospora khartoumensis, Gymnoascus gypseus,

Trichophyton spp, Trichosporon spp, and Zopfla rosatii.

[ 0082 ] A stimuli-responsive polysaccharide antimicrobial agent of the disclosure can be administered to any host, including a human or non-human animal, in an amount effective to inhibit the growth of a pathogenic microbe or microorganism. Thus, the stimuli- responsive polysaccharide antimicrobial agents disclosed herein are useful as broad-spectrum antimicrobials. Thus, the antimicrobial agents disclosed herein are suitable for tackling the growing problem of antibiotic-resistant bacteria strains, and for treating and/or preventing outbreaks of infectious diseases, including diseases caused by bioterrorism agents like anthrax, plague, cholera, gastroenteritis, multidrug-resistant tuberculosis (MDR TB). Any of a variety of art-known methods can be used to administer a stimuli-responsive polysaccharide antimicrobial agent disclosed herein either alone or in combination with one or more additional therapeutic agents. For example, administration can be parenterally, by injection or by gradual infusion over time. The stimuli-responsive polysaccharide antimicrobial agents alone or with other therapeutic agents can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, by inhalation, or transdermally.

[ 0083] The disclosure further provides a method for inhibiting a bacterial, viral and/or fungal-associated disorder by contacting or administering a therapeutically effective amount of a stimuli-responsive polysaccharide antimicrobial agent disclosed herein either alone or in combination with other antimicrobial agents to a subject who has, or is at risk of having, such a disorder. The term "inhibiting" means preventing or ameliorating a sign or symptoms of a disorder (e.g. , a rash, sore, and the like). Examples of disease signs that can be ameliorated include an increase in a subject's blood level of TNF, fever, hypotension, neutropenia, leukopenia, thrombocytopenia, disseminated intravascular coagulation, adult respiratory distress syndrome, shock, and organ failure. Examples of subjects who can be treated in the disclosure include those at risk for, or those suffering from, a toxemia, such as endotoxemia resulting from a gram-negative or gram-positive bacterial infection. Other examples include subjects having a dermatitis as well as those having skin infections or injuries subject to infection with gram-positive or gram-negative bacteria, a virus, or a fungus. Examples of candidate subjects include those suffering from infection by E. coli, Neisseria meningitides, staphylococci, or pneumococci. Other subjects include those suffering from gunshot wounds, renal or hepatic failure, trauma, bums, immunocompromising infections (e.g. , HIV infections), hematopoietic neoplasias, multiple myeloma, Castleman's disease or cardiac myxoma. Those skilled in the art of medicine can readily employ conventional criteria to identify appropriate subjects for treatment in accordance with the disclosure.

[ 0084 ] A therapeutically effective amount can be measured as the amount sufficient to decrease a subject's symptoms (e.g., high fever, pain, redness, soreness, etc.). Typically, the subject is treated with an amount of a stimuli-responsive polysaccharide antimicrobial agent disclosed herein that is sufficient to reduce a symptom of a microbial infection (e.g., a bacterial infection) by at least 50%, 90% or 100%. Generally, the optimal dosage will depend upon the disorder and factors such as the weight of the subject, the type of bacteria, virus or fungal infection, the weight, sex, and degree of symptoms. Nonetheless, suitable dosages can readily be determined by one skilled in the art. Typically, a suitable dosage is 1 to 1000 mg/kg body weight, e.g., 10 to 500 mg/kg body weight. In furtherance of any embodiment presented herein, a stimuli-responsive polysaccharide antimicrobial agent disclosed herein is administered at dosage of 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 110 mg/kg, 120 mg/kg, 130 mg/kg, 140 mg/kg, 150 mg/kg, 160 mg/kg, 170 mg/kg, 180 mg/kg, 190 mg/kg, 200 mg/kg, 210 mg/kg, 220 mg/kg, 230 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 100 mg/kg, or is a range that includes or is between any two of the foregoing dosages, including fractional dosages thereof.

[ 0085] As mentioned previously, the compositions and methods of disclosed herein can include the use of additional (e.g., in addition to a stimuli-responsive polysaccharide antimicrobial agent disclosed herein) therapeutic agents (e.g. , antibiotic, gene silencing molecules and the like). A stimuli-responsive polysaccharide antimicrobial agent disclosed herein, therapeutic agent(s), antifungal agents and/or antibiotic(s) can be administered simultaneously or sequentially. Suitable antibiotics include, but are not limited to, penicillins (e.g., penicillin V potassium, amoxicillin, amoxicillin/clavulanate (Augmentin)); tetracyclines (e.g., doxycycline, tetracycline, minocycline); cephalosporins (e.g. , cefuroxime, ceftriaxone, cefdinir); quinolones (e.g., ciprofloxacin, lev ofloxacin, moxifloxacin); lincomycins (e.g. , clindamycin, lincomycin); macrolides (e.g. , azithromycin, clarithromycin, erythromycin); sulfonamides (e.g., sulfamethoxazole-trimethoprim, sulfasalazine, sulfisoxazole);

glycopeptide antibiotics (e.g. , dalbavancin, oritavancin, telavancin, vancomycin);

aminoglycosides (e.g., gentamicin, tobramycin, amikacin); and carbapenems (e.g., imipenem/cilastatin, meropenem, doripenem, ertapenem). Suitable antifungal agents include, but are not limited to, azole antifungals (e.g., itraconazole, posaconazole, ketoconazole, clotrimazole, miconazole, voriconazole); echinocandins (e.g., caspofungin, anidulafungin, micafungin); polyenes (e.g. , nystatin, amphotericin b); and antimycotic agents (e.g. , griseofulvin, terbinafine, flucytosine). In furtherance of any embodiment presented herein, the stimuli-responsive polysaccharide antimicrobial agent disclosed herein is administered with one or more antibiotics including, but not limited to, Amikacin, Gentamicin,

Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Streptomycin,

Spectinomycin, Geldanamycin, Herbimycin, Rifaximin, Loracarbef, Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin, Cephradine, Cephapirin,

Lomefloxacin, Moxifloxacin, Nadifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,

Sulfonamidochrysoidine, Demeclocycline, Doxycycline, Metacycline, Minocycline,

Oxy tetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine,

Tinidazole, and/or Trimethoprim.

[ 0086] Generally, the antibiotic or antifungal agent is administered in amount to suppress or kill bacteria or fungi, respectively. Their effects can also be augmented by coadministration with an inhibitor of flavohemoglobin, (Helmick et al. , Imidazole antibiotics inhibit the nitric oxide dioxygenase function of microbial flavohemoglobin. Antimicrob Agents Chemother, 2005,49(5): 1837-43, and Sud et al, Action of antifungal imidazoles on Staphylococcus aureus, Antimicrob Agents Chemother, 1982, 22(3):470-4), increasing the efficacy of NO-based S. aureus killing by macrophages, and optionally triple combination therapies, may be applied to a patient in need of therapy. For example, a triple combination therapy can comprise a squalene synthase inhibitor, a flavohemoglobin (nitric oxide dioxygenase) inhibitor such as an azole (miconazole, econazole, clortrimazole, and ketoconazole) and a stimuli-responsive polysaccharide antimicrobial agent of the disclosure.

[ 0087 ] A pharmaceutical composition comprising a stimuli-responsive polysaccharide antimicrobial of the disclosure can be in a form suitable for administration to a subject using carriers, excipients, and additives or auxiliaries. Frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol, and polyhydric alcohols. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial, chelating agents, and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co., 1405-1412, 1461-1487 (1975), and The National Formulary XIV., 14th ed., Washington: American Pharmaceutical Association (1975), the contents of which are hereby incorporated by reference. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's, The Pharmacological Basis for Therapeutics (7th ed.).

[ 0088 ] The pharmaceutical compositions according to the disclosure may be administered locally or systemically. A "therapeutically effective dose" is the quantity of a stimuli-responsive polysaccharide antimicrobial according to the disclosure necessary to prevent, to cure, or at least partially arrest the symptoms of a microbial infection. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the subject. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of specific infections. Various considerations are described, e.g. , in Langer, Science, 249: 1527, (1990); Gilman et al. (eds.) (1990), each of which is herein incorporated by reference. [ 0089] As used herein, "administering a therapeutically effective amount" is intended to include methods of giving or applying a pharmaceutical composition of the disclosure to a subject that allow the composition to perform its intended therapeutic function, e.g., eliminate a microbial infection. The therapeutically effective amounts will vary according to factors, such as the degree of infection in a subject, the age, sex, and weight of the individual.

Dosage regime can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[ 0090 ] The disclosure further provides for a pharmaceutical composition comprising a stimuli-responsive polysaccharide antimicrobial agent(s) disclosed herein that can be administered in a convenient manner, such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the pharmaceutical composition can be coated with a material to protect the pharmaceutical composition from the action of enzymes, acids, and other natural conditions that may inactivate the pharmaceutical composition. The pharmaceutical composition can also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[ 0091 ] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size, in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be typical to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[ 0092 ] Sterile inj ectable solutions can be prepared by incorporating the

pharmaceutical composition in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the pharmaceutical composition into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.

[ 0093] The pharmaceutical composition can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The pharmaceutical composition and other ingredients can also be enclosed in a hard or soft-shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet. For oral therapeutic administration, the pharmaceutical composition can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations can, of course, be varied and can conveniently be between about 5% to about 80% of the weight of the unit.

[ 0094 ] The tablets, troches, pills, capsules, and the like can also contain the following: a binder, such as gum gragacanth, acacia, com starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent, such as com starch, potato starch, alginic acid, and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin, or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar, or both. A syrup or elixir can contain the agent, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic/biocompatible in the amounts employed. In addition, the pharmaceutical composition can be incorporated into sustained-release preparations and formulations.

[ 0095] The stimuli-responsive polysaccharide antimicrobial agent disclosed herein can be formulated either alone or in combination with other therapeutic agents (e.g. , antibiotics/antifungals) for topical administration (e.g., as a lotion, cream, spray, gel, or ointment). Such topical formulations are useful in treating or inhibiting microbial, fungal, and/or viral presence or infections on the eye, skin, and mucous membranes (e.g. , mouth, vagina). Examples of formulations in the market place include topical lotions, creams, soaps, wipes, and the like. It may be formulated into liposomes to reduce toxicity or increase bioavailability. Other methods for delivery include oral methods that entail encapsulation of the in microspheres or proteinoids, aerosol delivery (e.g., to the lungs), or transdermal delivery (e.g., by iontophoresis or transdermal electroporation). Other methods of administration will be known to those skilled in the art.

[ 0096] Also provided herein are preparations for parenteral administration of a composition comprising a stimuli-responsive polysaccharide antimicrobial agent of the disclosure include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Examples of aqueous carriers include water, saline, and buffered media, alcoholic/aqueous solutions, and emulsions or suspensions. Examples of parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives such as, other antimicrobial, anti-oxidants, cheating agents, inert gases and the like also can be included.

[ 0097 ] Thus, a "pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the pharmaceutical composition, use thereof in the therapeutic

compositions and methods of treatment is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[ 0098 ] It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein, refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of pharmaceutical composition is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are related to the characteristics of the pharmaceutical composition and the particular therapeutic effect to be achieve.

[ 0099 ] The principal pharmaceutical composition is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

[ 00100 ] An added advantage of the stimuli-responsive polysaccharide antimicrobial agents disclosed herein is the possible use of the agents to target intracellular infections. In particular, it was found that compositions comprising the stimuli-responsive polysaccharide antimicrobial agents disclosed herein that have been complexed or polyplexed with polymers can improve uptake by cells by various cellular mechanisms, e.g., phagocytosis, endocytosis, pinocytosis, or micropinocytosis. In a particular, it was found that compositions comprising the stimuli-responsive polysaccharide antimicrobial agents of the disclosure which have been complexed or polyplexed with polymers can provide for particles of various sizes. For example, particles of up 200 nm were made from the stimuli-responsive polysaccharide antimicrobial agents of the disclosure that were complexed or polyplexed with

poly deoxyribonucleic acid (pdrn). Moreover, these 200 nm particles were efficiently internalized by macrophages via phagocytosis. In furtherance of any embodiment presented herein, the disclosure provides for particles or similar type structures that comprise a composition the stimuli-responsive polysaccharide antimicrobial agents of the disclosure that have been complexed or polyplexed with a polymer, like pdrn or other nucleic acid. In further embodiment, the particles comprising said compositions have diameters of 1 nm, 2 nm, 5nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, or is a range that includes or is between any two of the foregoing values, including factional values thereof.

[ 00101 ] Intracellular infections are usually the most difficult to target due to intracellular diffusion kinetics and lack of drug accessibility to desired locations. Unlike other nanoantibiotic materials, it is postulated that the antimicrobial agents of the disclosure would be very effective against such intracellularly located microbes (e.g. , intracellular bacteria), without exhibiting the toxicity to host cells seen with other nanoantibiotic materials. Intracellular bacteria can be divided into two groups, facultative intracellular bacteria and obligate intracellular bacteria. Facultative intracellular bacteria invade host cells when it gives them selective advantage in the host. Bacteria that can enter and survive within eukaryotic cells are shielded from humoral antibodies and can be eliminated only by a cellular immune response. Most of these bacteria must possess specialized mechanisms to protect them from the harsh environment of the lysosomal enzymes encountered within the cells. For example, Legionella pneumophila prefers intracellular environment of

macrophages for growth so it induce its own uptake and blocks lysosomal fusion by undefined mechanism; R rickettsii destroys the phagosomal membrane with which the lysosomes fuse; Mycobacterium tuberculosis inhibits phagosome-lysosome fusion; Listeria monocyotogenes quickly escapes the phagosome into the cytoplasm before phagosome- lysosome fusion; Salmonella spp is very resistant to intracellular killing by phagocytic cells. Other facultative intracellular bacteria include, but are not limited to, Escherichia coli, Neisseria spp, Brucella spp, Shigella spp. In regards to obligate intracellular bacteria, this group of bacteria can't live outside the host cells. For example, Chlamydial cells are unable to carry out energy metabolism and lack many biosynthetic pathways, therefore they are entirely dependent on the host cell to supply them with ATP and other intermediates. Other obligate intracellular bacteria include, but are not limited to, Mycobacterium leprae, Coxiella burnetii, and Ricekettsia spp. In furtherance of any embodiment presented herein, the stimuli-responsive polysaccharide antimicrobial agents disclosed herein are used to treat an intracellular bacterial infection is caused by bacterium selected from the genus

Chlamydophila, Ehrlichia, Rickettsia, Chlamydia, Salmonella, Neisseria, Brucella,

Mycobacterium, Nocardia, Listeria, Francisella, Legionella, or Yersinia pestis.

[ 00102 ] It was further found herein that the stimuli-responsive polysaccharide antimicrobial agents disclosed herein can also serve as vectors for delivering, e.g., siRNAs or other gene silencing molecules, into the interior of cells. Thus, the stimuli-responsive polysaccharide antimicrobial agents of the disclosure are envisaged as being multifactorial agents that can not only be used as antimicrobial agents by themselves but can further be used as vectors to deliver additional therapeutic agents intracellularly. Moreover, the additional therapeutic agents can be triggered to release from the stimuli-responsive polysaccharide antimicrobial agents of the disclosure based upon a change in pH, such as exposure to the low pH environment of a lysosome (e.g. , see FIG. 15). In furtherance thereof, it was found that the stimuli-responsive polysaccharide antimicrobial agents disclosed herein comprising ketal linkages that were cleavable in the mildly acidic endosome/lysosome greatly aided endosomal escape of siRNA via dissociation from hydrolyzed the stimuli-responsive polysaccharide antimicrobial agents and increased osmotic pressure via release of side branches. Acid hydrolyzed stimuli-responsive polysaccharide antimicrobial agents self- aggregate with low solubility at a neutral pH and releases free siRNA for binding to the RISC complex. The acid-transformation of the stimuli-responsive polysaccharide antimicrobial agents disclosed herein in the endosome/lysosome to native chitosan makes it a promising carrier in treating intracellular infections. Accordingly, the stimuli-responsive polysaccharide antimicrobial agents of the disclosure directly provide an antimicrobial effect on microbes upon conversion to native chitosan, but further is capable of delivering antimicrobial agents such as antibiotics and nucleic acids. For purpose of this disclosure, "nucleic acids" includes polydeoxyribonucleotide (pdm), mRNA, rRNA, tRNA, RNAase P, small nuclear RNA (snRNA), CRISPR RNAs (crRNAs), gRNA, small nucleolar RNA (snoRNA), efference RNA (eRNA), tmRNA, small interfering RNA (siRNA), small non-coding microRNA (miRNA), ssRNA, repeat associated small interfering RNA (rasiRNA), piwi-interacting RNA (piRNA), antisense RNA (asRNA), DNA, and cDNA. For example, the stimuli-responsive polysaccharide antimicrobial agents disclosed herein can be complexed or polyplexed with asRNA that targets antibiotic resistance genes or asRNA that targets genes associated with bacterial or fungal growth, a CRISPR-Cas system (e.g. , Cas-9 or Cas-3), a CRISPRi system or a CRISPR-Cpfl system that targets antibiotic resistance genes (e.g. , see Reardon, Nature 546(7660):586-587) or a CRISPR-Cas system, a CRISPRi system or a CRISPR-Cpfl system that targets genes associated with bacterial or fungal growth (e.g. , Citorik et al, Nature Biotechnology 32: 1141-1145 (2014)), and/or siRNA (e.g. , siRNA that targets genes that are involved in the immune response caused by a fungal or bacterial infection, or siRNA that targets host genes involved in mediating the entry of bacteria or fungus into host cells but also inhibits the growth of or destroys microbes upon acid-transformation.

[ 00103] The relatively large size of the stimuli-responsive polysaccharide

antimicrobial agents/nucleic acid polyplexes could be particularly suitable for treating microbial infections (e.g. , salmonella) in phagocytic cells (e.g., macrophages). In furtherance of any embodiment presented herein, the antibacterial agent disclosed herein is used an antibacterial agent and as a vector to administer one or more additional therapeutic agents. In furtherance of any embodiment presented herein, the one or more additional therapeutic agents are siRNA, small non-coding microRNAs, or asRNAs. Designs, considerations and targets for asRNA and/or small non-coding microRNA in inhibiting of expression of pathogenic bacteria include those described in Good et al. {Front Microbiol, 2: 185 (2011)), and references cited therein. Particular gene targets can include genes that are required for bacteria or fungal replication and growth, and genes that impart antibiotic resistance to bacteria. In furtherance of any embodiment presented herein, the disclosure provides for a composition which comprises a stimuli-responsive polysaccharide antimicrobial agent that is complexed or polyplexed with a CRISPR-Cas system, a CRISPRi system or a CRISPR-Cpfl system that is used to target microbial genes that are involved with microbial growth, antibiotic resistance, or are necessary for the microbe's survival (e.g. , the CRISPR system can target a microbe gene that intrudes a mutation or deletion that results in the death of the microbe).

[ 00104 ] Additionally, ATC can be used to coat the hydrophilic surface of implantable devices for aseptic applications. The ketal linker and the aminoethoxy branch could be replaced by other stimuli-responsive linkers and side chains as has been indicated herein, depending on specific demands by other biomedical applications.

[ 00105 ] The following examples are intended to illustrate but not limit the disclosure. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used.

EXAMPLES

[ 00106 ] Materials. All chemicals were purchased from commercially available sources and used as received. Chitosan (MW 18-44 kDa, 80% degree of deacetylation), branched polyethylenimine (PEI) (MW 25 kDa), and 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyltetrazolium bromide (MTT) were purchased from Sigma- Aldrich (Milwaukee, WI). 2,2,2-Trifluoro-l-{2-[2-(l-methoxy-l-methylethoxy)ethoxy] ethylamino}-l-ethanone (trifluoroacetate [TF A] -protected aminoethoxy branch with ketal linkage, TFA-AE-k) was synthesized as previously reported in Kwon (2011) et al. (J. Controlled Release

150(3):287-297 (2011)) md Kwon (2009) et al. (Bioconjugate Chem. 20(3):488-499 (2009)). Phthalic anhydride and hydrazine monohydrate were purchased from Acros Organics (Morris Plains, NJ).

[ 00107 ] HeLa cells (ATCC, Rockville, MD) were cultured in Dulbecco's modified Eagle's medium (DMEM) (MediaTech, Herndon, VA) with 10% fetal bovine serum (FBS) (Hy clone, Logan, UT) and 1% antibiotics (100 units/mL penicillin; 100 μg/mL streptomycin) (MediaTech, Herndon, VA). HeLa cells stably expressing GFP (HeLa/EGFP) were prepared by transducing them with retrovirus encoding enhanced green fluorescence protein (eGFP) and further sorting them by FACS, as described for the preparation of NIH 3T3/EGFP cells.

[ 00108 ] General Synthesis Strategy for Acid-transforming chitosan. A scheme for synthesizing acid-transforming chitosan (ATC) is presented in FIG. 9. As shown, chitosan is reacted with phthalic anhydride in a solvent system comprising dimethyl formamide and 5% H2O at 120 °C to afford phthalimide-protected chitosan (Phth-C). After the reaction is completed, Phth-C is reacted with a trifluoroacetamide-protected aminoethoxy ketal linker (TFA-AE-k) in tetrahydrofuran at ambient temperature to form Phth- & TFA-protected chitosan (Phth-TFA-C). After which, Phth-TFA-C is first treated with 1M NaOH in H2O at ambient temperature and then treated with 20% hydrazine in H2O at 90 °C to afford the titled product.

[ 00109] ATC Synthesis.

[ 00110 ] Phthalimide-Protected Chitosan (Phth-C): Chitosan (1.0 g, 6.0 mmol pyranose) and phthalic anhydride (4.0 g, 20.0 mmol) were dissolved in 30 mL of 5% (v/v) deionized water in DMF and heated to 120 °C under nitrogen for 7 h. The reaction was stopped by mixing with 300 mL of deionized water. Phth-C was obtained after vacuum filtration in a fritted funnel and then dried under vacuum for 4 h at room temperature. Yield: 90%. FT-IR (KBr, cm ¹): 2950-2800 (alkyl), 1777-1670 (carbonyl), 1250-950 (pyranose), 728 (arom). ¾ NMR (500 MHz, DMSO-de, δ, ppm): 1.9 (m, acetyl), 3.0-5.2 (m, pyranose), 7.5-7.9 (m, N-phthaloyl). MALDI-TOF MS (m/z): 18 000-44 000.

[ 00111 ] Phth-C Conjugated with TFA-Protected Aminoethoxy Branches via Ketal Linkage (Phth-TFA-AE-k-C) . Phth-C (1.0 g, 1.91 mmol), pyridinium p-toluenesulfonate (PPTS) (2.0 g, 7.96 mmol), and TFAAE-k (3.0 g, 10.0 mmol) were mixed in 20 mL of anhydrous THF and 5 A sieves (10 g) for 3 h at 25 °C, then the reaction was quenched by adding 5 mL of triethylamine (TEA). Residual PPTS and unreacted TFA-AE-k were removed by repeated rinsing three times with 100 mL of methanol (MeOH), then dried under vacuum for 4 h at room temperature, resulting in Phth-TFA-AE-k-C as tan-brown solid. Yield: 40%. FT-IR (KBr, cm ¹): 2950-2800 (alkyl), 1777-1670 (carbonyl), 1200-1000 (pyranose), 728-700 (arom). ¾ NMR (500 MHz, DMSO-d6, δ, ppm): 1.3 (s, ketal), 1.9 (s, acetyl), 3.0-5.2 (m, pyranose), 7.5-7.9 (m, N-phthaloyl), 9.5 (s, acetamide). MALDI-TOF MS(m/z): 18 000-51 000.

[ 00112 ] Chitosan Conjugated with Aminoethoxy Branches via Ketal Linkage (A TC) . Phth-TFA-AE-C (1.0 g, 1.0 mmol) was added to 10 mL of 1 M NaOH and stirred at room temperature for 24 h for TFA de-protection. After the precipitate was removed by centrifugation, the liquid fraction was added to 100 mL of 20% (v/v) hydrazine in deionized water and stirred at 90 °C for 16 h for Phth-deprotection. The final product, ATC as white- colored powder, was obtained after dialysis in deionized water for 24 h and freeze-drying (Freezone 2L, Labconco [Kansas City, MO]) for 18 h. Yield: 50%. FT-IR (KBr, cnTl): 2950-2800 (alkyl), 1665-1650 (carbonyl), 1200-900 (pyranose). ¾ NMR (500 MHz, D₂0, δ, ppm): 1.46 (s, ketal), 2.1 (s, acetyl), 3.3-4.0 (m, pyranose). MALDI-TOF MS (m/z): 11 000-50 000.

[ 00113] Chemical Characterization of ATC and Chitosan. Proton nuclear magnetic resonance (¾ NMR) was recorded on Bruker (Billerica, MA) DRX500 spectrometer with a BBO probe as standard with 10 mg of ATC and chitosan samples in 1 mL of DMSO-d6 or D2O/DCL Fourier transform infrared (FT-IR) spectra were obtained on Jasco 4700 spectrophotometer (Oklahoma City, OK) between 4000 and 400 cm^"1 with a resolution of 4 crrT¹ on pressed 1% (w/w) ATC and chitosan samples in potassium bromide (KBr) windows. Matrix-assisted laser desorption/ionization (MALDI)-TOF measurements were completed on AB SCIEX (Redwood City, CA) TOF/TOF 5800 System with 10 mg/mL 2,5- dihydroxybenzoic acid in 50% (v/v) acetonitrile in deionized water (1% trifluoracetic acid) as a matrix solution. The solubility of ATC and chitosan were observed by dissolving them in pH 5.0 acetate buffer (100 mM) at 37 °C for 4 h or DI water at a concentration of 10 mg/mL. ATC and chitosan solutions in pH 5.0 acetate buffer (1 mL) were then neutralized with 200 of NaOH (1 M in DI water) to show reduced ATC to native chitosan with lowered solubility. In order to quantify the solubilized chitosan and ATC, the solutions were centrifuged at 4000 rpm for 5 min to remove any precipitates, followed by measuring the absorbance of 10 of supernatants at 290 nm wavelength using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

[ 00114 ] Preparation of ATC/siRNA Polyplexes. Desired amounts of ATC in 100 \L of DI water was dropwise mixed with eGFP siRNA (1.5 μg) in 100 μΐ. of DI water to yield various N/P ratios (molecular ratios of ATC's nitrogens to siRNA's phosphates), followed by a brief vortex and incubation at room temperature for 30 min to form ATC/siRNA polyplexes. The resulting polyplex solution was diluted with an additional 800 μΐ. of DI water. Mean particle diameter (Z-average) with a polydispersity index (PDI) and zeta potential of various ATC/siRNA polyplexes were measured by dynamic light scattering (DLS) particle analysis using a Zetasizer Nano ZS (Malvern, UK) at 25 °C and angle of 90°. The viscosity (0.887 mPA/s) and refractive index (1.33) of water at 25 °C were used to analyze the data. PEI (8 μg) in 150 of DI water was dropwise mixed with eGFP siRNA (1.5 μg) in 150 of DI water, followed by a brief vortex and incubation at room temperature for 30 min to form PEI/siRNA polyplexes at N/P 10.

[ 00115] Assays for siRNA Complexation by ATC. The ability of ATC to complex siRNA was evaluated by a standard ethidium bromide (EtBr) exclusion assay. EtBr (1 μg) and siRNA (1 μg) were incubated in 40 μΐ. of DI water for 15 min at room temperature. Desired amounts of ATC solution in 60 μΐ. of DI water were added to achieve various N/P ratios and vortexed. After 30 min of incubation at room temperature, fluorescence intensity (λβχ = 320 nm and em = 600 nm) was measured using a fluorescence microplate reader (Synergy HT, BioTEK [Winooski, VT]). Reduced fluorescence intensity was used as a quantitative indicator of siRNA condensation in the polyplexes.

[ 00116] The ATC's siRNA complexation efficiency was also determined by agarose gel electrophoresis. Desired amounts of ATC dissolved in 100 μΐ. of DI water were vortexed with 1 μg of siRNA in 100 μΐ. of DI water to yield polyplexes at various N/P ratios.

Polyplexes (18 μΐ. mixed with 2 μΐ. DNA loading dye [Thermo Fisher Scientific, Waltham, MA]) were loaded into each well of 1% (w/v) agarose gel.

[ 00117 ] To demonstrate the dissociation of the siRNA from ATC upon acid transformation, the polyplexes were mixed with an equal volume of pH 5.0 acetate buffer (100 mM acetic in DI water adjusted by 50 mM sodium acetate trihydrate) and further incubated for 4 h at 37 °C. The acid-transformation was quenched by adding 5 μί of 1 N NaOH into 20 μΐ_^ samples and incubating the mixture for another 15 min. An 18 μΐ_^ aliquot of the samples was mixed with 2 μΐ. of loading dye, and the resulting mixture solution was loaded on the agarose gel. Electrophoresis was carried out at a constant voltage of 45 V for 10 min then 100 V for 30 min in Tris-acetate-EDTA (TAE) buffer. Bands were then visualized under a UVP transilluminator (Analytik, Jena, Germany) at a wavelength of 365 nm.

[ 00118 ] Morphology of ATC/siRNA Polyplexes. The morphology of various polyplexes was analyzed by transmission electron microscopy (TEM). ATC/siRNA polyplexes at varying N/P ratios prepared as described earlier in 10 μί deionized water were dropped on a hydrophobic wax paper, then a carbon-coated copper TEM grid (Electron Microscopy Sciences [Hatfield, PA]) was placed on the sample droplet for 10 min at room temperature. The grid was then air dried for 2 h at room temperature. The grids were imaged with a Philips/FEI (Hillsboro, OR) CM-20 Transmission Electron Microscope operated at 200 kV.

[ 00119] Silenced eGFP Expression in HeLa Cells by A TC siRNA Polyplexes.

HeLa/eGFP cells were seeded overnight in a 24-well plate at a density of 20,000 cells/well. Prior to transfection, culture media was removed, and cells were washed once with PBS. The ATC/siRNA polyplexes were prepared as described earlier using eGFP siRNA or scrambled (scr) siRNA solutions in PBS instead of DI water at a concentration of 80 μΜ. There were no significant differences between the polyplexes prepared with eGFP siRNA and scr siRNA in size, zeta potential, and PDI. Following 30 min of incubation, polyplex solutions (100 in PBS) were diluted with 300 μΐ. of FBS-free DMEM before it was added to the wells. The cells were incubated for 4 h with ATC/siRNA polyplexes at 37 °C with a 5% CC

atmosphere. After 4 h, the polyplex-containing medium was aspirated, and the cells were rinsed with PBS once, followed by the addition of 400 μΐ, of DMEM with 10% FBS and further incubation for 72 h. Fluorescence imaging and flow cytometry (Guava Easycyte Plus, [MilliporeSigma, Burlington, MA]) were performed to confirm eGFP silencing in the cells.

[ 00120 ] ATC Synthesis and Molecular Characterization. Chitosan was conjugated with acid cleavable, cationic side chains to synthesize acid-transforming chitosan (ATC) with improved aqueous solubility, and to provide for the release chitosan into the cytoplasm upon acid hydrolysis of the ketal linkage in the mildly acidic endosome (see FIG. 9). Successful ATC synthesis was confirmed by the peak at 1.4 ppm, indicative of the dimethyl ketal linkage, and disappearance of the peaks for the phthalimide and TFA groups at 7.8 and 9.5 ppm, respectively, in ¾ NMR spectra (see FIG. 10A). Comparison of the dimethyl ketal (1.4 ppm) with pyranose (3.5-3.8 ppm) peak integrations determined that approximately 13% of primary hydroxyl groups were conjugated with the acid-cleavable, cationic side chain. FTIR also confirmed the alkyl (yellow), carbonyl (blue), pyranose (pink), and aromatic (green) groups (see FIG. 10B). The peaks representing carbonyl, pyranose, and aromatic groups exhibited noticeable changes at the protected intermediate, but no visible peak shifts were observed in the alkyl region. Chitosan has stronger CFh and CFb vibrations than the conjugated aminoethoxy branch, which could have caused the lack of significant vibrational change in the alkyl region.

[ 00121 ] Molecular characterizations of chitosan offer significant technical challenges primarily due to its poor aqueous solubility. While chitosan requires an acidic solvent, ATC should avoid an acidic solvent for characterization. Therefore, it was important to use the combination of multiple molecular characterization methods such as ¾ NMR, FTIR, and MALDITOF in characterizing ATC and its intermediates (see FIG. 10). MALDI-TOF provided an estimated range of the molecular weight of chitosan, the intermediate, and ATC (see FIG. IOC). The dashed line was used to indicate the peak of the curvature. Between chitosan and phth-TFA-C, the peak shifted from 28 000 to 32 000 m/z. The shift was attributed to conjugation of TFA-AE-k to chitosan as confirmed by conjugation efficiency (i.e. , ~13%) as calculated by ¾ NMR. After removal of the phth and TFA protecting groups, the MALDI-TOF peak shifted back to 27,000 m/z. The average molecular weight of ATC was lower than chitosan, but the molecular weight distribution of ATC (11 000-50 000 m/z) was wider than that of chitosan (18 000-44 000 m/z). This could be attributed by significant differences between chitosan and ATC in solubility for separation and purification during synthesis as well as sample preparation for characterization. The molecular weights estimated by MALDI-TOF could be complemented by other methods such as gel permeation chromatography (GPC) and provide crucial information for understanding and predicting its molecular behaviors and interactions with nucleic acids; however, ATC and its intermediates tend to aggregate in a column, interfering with obtaining an accurate molecular weight measurement.

[ 00122 ] Acid-Transformation of ATC to Native Chitosan. Acid triggered ATC transformation to chitosan was demonstrated by changes in molecular structure and solubility at various pHs (see FIG. 11). After incubation at pH 5.0 for 16 h, the peaks for the ketal linkage (1.4 ppm, q, r) disappeared, the broad peak at 3.5-4.0 ppm (u-w) was reduced to two distinct peaks upon the loss of the aminoethoxy side chain, and a sharp peak at 2.2 ppm for acetone, a byproduct of ketal hydrolysis, appeared (see FIG. 11A). Acid-transformation of ATC to chitosan was also obvious based upon solubility changes (see FIG. 11B). While chitosan rapidly precipitates in water, ATC was instantaneously and fully dissolved in water. At an endosomal pH of 5.0, chitosan became soluble but precipitated severely upon neutralization, while hydrolyzed ATC at pH 5.0 became insoluble without visible precipitation upon neutralization (see FIG. 12). The half-lives of ATC at pH 5.0, 6.0, and 7.4 were calculated to be 0.48, 0.64, and 18 h, respectively, while the half-lives of ATC/siRNA polyplexes at the same pHs were 27, 29, and 99 h, respectively (See FIG. 13). This indicates that the reduced water accessibility to the electrostatically attracted ATC to siRNA and that the anionic siRNA greatly reduced the acid-hydrolysis of ATC. Neutralization of acid- incubated ATC reduced its solubility due to acid hydrolysis of ketal linkages of aminoethoxy side chains. This observation indicates molecular changes of ATC during extracellular and intracellular processes; ATC is water-soluble during extracellular transport and cellular uptake, gets acid hydrolyzed in the endosome/lysosome and undergoes transformation, and is released as chitosan into the cytoplasm. Interestingly, ATC solution incubated at pH 5.0 and further neutralized became a homogeneous suspension of small particulates (575 ± 42 nm), while much larger aggregates (1110 ± 425 nm) of chitosan treated in the same way (see FIG. 12). This observation implies faster clearance of hydrolyzed ATC by the reticuloendothelial system (RES) and enzymatic degradation as well as lower risk of causing adverse effects such as embolism than native chitosan. The results shown in FIG. 11 implicate efficient siRNA complexation by ATC with improved aqueous solubility and molecular interactions with cationic side chains, rapid siRNA release upon acid hydrolysis of cationic side chains in the mildly acidic endosome/lysosome and avoided re-complexation of siRNA in the cytoplasm by self-aggregated, hydrolyzed ATC.

[ 00123] siRNA Complexation by ATC and the Morphology of siRNA/ A TC

Polyplexes. Since nucleic acids are anionic due to the charged backbone, the improved aqueous solubility and flexible cationic side chains of ATC were speculated to contribute to efficient siRNA complexation via enhanced electrostatically attractive molecular interactions. ATC complexed siRNA and formed ATC/siRNA polyplexes in a size range from 180 to 400 nm in an N/P ratio range of 10-100 within a consistent PDI of approximately 0.2 (see FIG. 14A). The zeta-potential transitioned from negative to positive between N/P ratios of 20 and 50. The unexpectedly low PDI at N/P 20 possibly indicates minimum numbers of uncomplexed ATC or siRNA (i.e. , excess siRNA or ATC at N/P < 20 or NP > 20, respectively). This indicates a molecular ratio of ATC to siRNA was approaching the point where both molecules complement each other by attractive electrostatic interactions without shortage or excess between N/P 20 and 50. As expected, this is higher than the optimal N/P ratios known for complexing nucleic acids by PEI (i.e. , N/P ratios of 5-10) due to the significantly lower cationic density of ATC than PEI. ATC has a total of two primary amines found on the pyranose backbone and another at the tip of the aminoethoxy branch per repeating unit (MW 338 Da), while branched PEI has a total of four primary amines per repeating unit (MW 401 Da). In addition, the pyranose backbone of ATC makes it less flexible than PEI. Altogether, the electrostatic interactions with siRNA by ATC and PEI are different even for the same N/P ratios. The zeta potential of ATC/siRNA polyplexes became positive at N/P ratio of 50 and further increased at N/P of 100 (see FIG. 14A) or higher due to the significant excess of positive charges provided by ATC for a given number of negative charges of siRNA. Ethidium bromide (EtBr) exclusion assay (see FIG. 14B) showed the reduction in fluorescence intensity as ATC shielded siRNA at increasing N/P ratio as anticipated. The shielding of siRNA by ATC was similar to those reported for chitosan, PEI, and PLL. siRNA complexation by ATC was further qualitatively assessed by agarose gel electrophoresis (see FIG. 14C). ATC complexed siRNA via the increased charge density and close interaction with the cationic aminoethoxy branches more effectively than chitosan and was able to release siRNA upon acid hydrolysis. Due to the significantly slower hydrolysis in ATC/siRNA polyplexes than free ATC (see FIG. 13), more siRNA was retained by ATC at high N/P ratios even at pH 5.0 (see FIG. 14C). TEM showed consistent spherical morphology of ATC/siRNA polyplexes (see FIG. 14D) and their sizes increased as N/P ratios increased (see FIG. 14A and 14D). Efficient delivery is dependent on the ability to efficiently protect siRNA, aid in cellular uptake, and ensure cytosolic release. ATC demonstrated the capability of effectively complexing siRNA and are suitable for transfecting phagocytic cells, particularly in vivo. However, polyplexes in a similar size have also been reported to efficiently transfect many kinds of cells in vitro as well as in vivo. One advantage of transfecting larger gene carriers is to deliver a large number of nucleic acids to a cell which seems to be important for gene silencing.

[ 00124 ] Efficiently Silenced eGFP Expression by ATC/siRNA Polyplexes with Low Cytotoxicity. The capability of ATC/siRNA polyplexes to silence the expression of a target gene was investigated by incubating them with HeLa cells expressing eGFP as a model gene. Polyplexes (20 μΜ) prepared at N/P ratio of 50 efficiently silenced eGFP expression from 85% to 0.7% without noticeable cytotoxicity after 72 h incubation, while eGFP siRNA alone or ATC/scr siRNA polyplexes did not affect eGFP expression (see FIG. 15). The PEI/siRNA polyplexes (N/P 10) showed moderate gene efficiency (~45%). The polyplexes prepared at a higher N/P ratio also showed significant eGFP silencing: expression decreased from 85% to 0.67% at N/P ratio of 100. However, this was accompanied by moderate cytotoxicity as well as measurable nonspecific gene silencing of 4.8%. ATC itself was nontoxic even at high concentrations (up to 1 mg/mL), in contrast to PEI showing high toxicity at much lower concentrations.

[ 00125] Antimicrobial properties of Acid-transforming chitosan (ATC), hydrolyzed ATC and chitosan on gram negative bacteria. In order to study the antimicrobial action of chitosan and acid-transforming chitosan (ATC) against gram negative bacteria, a time course study was performed in E. Coli using three different concentrations of chitosan or ATC (0.7 mg/mL, 3.5 mg/mL, and 7 mg/mL). As shown in FIG. 19, chitosan was much faster acting than ATC in killing E. Coli. Most of the bacteria were destroyed as early as 1 h when exposed to chitosan, while it took up to 4 h before significant reduction in colonies were seen when 3.5 mg/mL of ATC was used. However, colony growth was significantly depressed at 2 h when 7.0 mg/mL of ATC was used.

[ 00126] In direct contrast, hydrolyzed ATC showed similar early time point efficacy as chitosan. Hydrolyzed ATC exerted its antibacterial effects at 0.7 mg/mL and reduced the growth of bacteria after 16 h. Furthermore, the killing curve of hydrolyzed ATC resembled closely that of chitosan, and almost all the bacteria were dead after 2 hours when hydrolyzed ATC was used at 3.5 mg/mL and 7.0 mg/mL.

[ 00127 ] Toxicity of Acid-transforming chitosan (A TC) and chitosan in vitro on mammalian cell lines. The toxicity of ATC and chitosan were studied by using MTT assays in two mammalian cell lines, human HeLa cells and mouse Raw 264 cells. As shown in FIG. 20, it was found that ATC is much less toxic than chitosan regardless of the attempt or the cell type. At concentrations of 3.5 mg/mL or 7 mg/mL both polymers appear to be toxic to the cells. Raw 264.7 cells are more resistant to polymer toxicity, possibly due to

phagocytosis of macrophages. While the relative viability of ATC remains relatively constant throughout, the viability of chitosan varies a bit with each attempt. This might be due to various reasons, such as the varied sizes of chitosan polymer or just possible effect of the solubility.

[ 00128 ] Toxicity of Acid-transforming chitosan (A TC) and chitosan in vivo in C57BL/6 mice. Mice were administered various doses of chitosan or ATC and monitored over a period of twenty days. As shown in FIG. 21, it was found that mice who were injected with native chitosan at varied doses except 25 mg/kg died within 8 days post administration. Mice injected with 200 mg/kg chitosan died instantly, while the remaining mice died after an extended period of time. In direct contrast, only the mice who were injected with 200 mg/kg ATC died within the observed time period. Mice who received lower doses of ATC appeared to be healthy and well over the 20-day observation period.

[ 00129] Antimicrobial efficacy of ATC and toxicity of ATC. Intracellular pathogens can localize in cellular compartments such as early endosome, late endosome, or vacuoles, thereby protecting the pathogens from normal host cell defense. Each of these compartments has a pH ranging from 5.3 or lower for the late endosome to 7.4 for vacuoles. S. typhimurium, has been shown to transition from the late endosome to a specialized vacuole within the host cell. Therefore, in engineering an antimicrobial gene carrier against intracellular microbes like S. typhimurium, testing antimicrobial efficacy under varying pH conditions is indicated. To this end, the antimicrobial efficacy of ATC against S.

typhimurium at various pHs was confirmed prior to intracellular infection testing (see FIG. 22A). S. typhimurium was incubated with ATC and 25 mM of buffer (pH 5.5, 6, or 7) for 24 h (see FIG. 22A). Irrespective of the pH, less than 50% of microbes survived at an ATC concentration greater than 500 μg/mL. At pH 5.5, the percent of surviving microbes was decreased for all ATC concentrations, with 50 and 100 μg/mL of ATC being the most significant. The antimicrobial efficacy of chitosan was found to increase in an acidic pH. It was expected that ATC would function in the same manner. As the primary amines of chitosan have a pKa value of - 6.5, they are protonated in an acidic environment. The protonated amines are believed to specifically favor interactions with the wall and membrane of the microbes. As such, the damage to mammalian membranes is limited or nonexistent.

[ 00130 ] In vitro and in vivo toxicity is of high importance for the design of a nanoantibiotic platforms using ATC. Further, since intracellular bacteria are found within a mammalian cell, ATC needs to maintain minimal toxicity to the host but still effectively treat the infection. From the MTT assay of ATC against RAW 264.7 macrophage cells, no toxicity was observed at any of the tested concentrations (see FIG. 22B). No in vitro cellular toxicity was reported for ATC or unmodified chitosan. However, for in vivo studies, 50 mg/kg doses and higher of chitosan caused death (see FIG. 22C). The observed in vivo toxicity agreed with previous reports of the intravenous administration of chitosan. Blood aggregation was speculated as the cause of death. Interestingly, although ATC was derived from chitosan, in vivo toxicity was not observed at similar dosages. This reduced toxicity likely results from ATC being a water-soluble variant of chitosan. As such, ATC is less likely to cause blood aggregation upon intravenous administration. In the design of the platform, limiting acute toxicity was given greater significance since the focus was on the immediate eradication of pathogenic bacteria. However, possible long-term toxicities associated with chitosan and hence ATC, should be noted as well. While toxicities stemming from chronic use are only just starting to be explored, these toxicities are usually dose and concentration dependent. Some early studies have indicated some chronic in vivo toxicity in zebrafish models at dosages of 250 mg/L which is close to the lethal dose of 280 mg/L. While these chronic toxicities may seem worrisome, the toxicity only results from using chitosan at doses much higher than clinical relevance.

[ 00131 ] Formation of ATC/pdm polyplexes. As noted above, ATC displayed excellent antimicrobial activity against microbes in the extracellular context. It was expected, however, that ATC in a noncomplexed form, would have a hard time traversing the cell membrane. Accordingly, a polyplex of ATC with polydeoxyribonucleotide (pdrn) was generated so as to form particles around 200 nm in diameter (see FIG. 23). Pdrn was selected because it is comprised largely of negatively charged nucleic acids and lacks genetic specificity by not coding for any specific proteins. Thus, a polyplex of pdrn with ATC can be simply formed through electrostatic interaction. Polyplexes were created at an N/P ratio of 100 and as highly monodisperse particles with a PDI less than 0.1 (see FIG. 23). These particles also had a zeta potential of +13 mV. ATC/pdrn polyplexes were created and used in vitro without any additional purification. For in vivo studies and future clinical applications, further purification can be brought about by use of column chromatography to remove excess pdrn or ATC.

[ 00132 ] Utilization of A TC/pdrn polyplexes to treat intracellular infection.

Intracellular infections can occur in various cell types but is usually more prevalent in phagocytic cells. Therefore, RAW 264.7 cells were used. Furthermore, in most real-world applications, antibiotics are not used until the infection has been prolonged. ATC polyplexes were applied to prolonged infections to highlight the versatility of ATC as a nanoantibiotic. Therefore, to demonstrate an early infection, where there were minimal bacteria per cell, RAW 264.7 cells were infected with GFP expressing S. typhimurium for 1 h and then treated with ATC/pdrn polyplexes. Microbe death was confirmed using fluorescence microscopy and flow cytometry. For early-stage infections, the most significant results occurred at 500 and 1000 μg/mL of ATC/pdrn polyplexes (see FIG. 24). Bacterial growth was almost completely suppressed when the ATC/pdrn polyplexes were used at concentrations of 1000 μg/mL. This result correlates well with the antimicrobial efficacy observed with uncomplexed ATC. For a prolonged intracellular infection, RAW 264.7 cells were infected with GFP expressing S. typhimurium that were allowed to grow for 16 h before ATC/pdrn polyplexes were introduced (see FIG. 25). There was significant reduction of up to 40% in bacteria as the concentration of polyplex increased. Even at low concentrations of polyplex, 100 mg/mL, there was a 9% reduction in the median fluorescence intensity compared to the control.

Though this was a single dose study, continued treatment could yield a further reduction in bacteria growth. The antimicrobial capacity against an early-stage intracellular infection was further emphasized in cell lysis data (see FIG. 26). Bacteria at 1000 μg/mL of ATC were almost completely eliminated, and intracellular survival of colonies was almost negligible. Based upon the colony count, 100 μg/mL polyplexes reduced S. typhimurium growth by twofold compared to control. The colony count data reflected a larger antibacterial effect than was observed in the flow cytometry data and fluorescence images. It is possible that there were false positive fluorescent readings attributed to extracellular bacteria, since it was expected that the highest antimicrobial activity would occur intracellularly.

[ 00133] It will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A stimuli-responsive polysaccharide antimicrobial agent that comprises the structure of Formula I:

Formula I

or a pharmaceutically acceptable salt thereof,

wherein,

AR¹ is an acid labile group that can be cleaved or hydrolyzed when exposed to a mildly acidic environment;

AR² is an acid labile group that can be cleaved or hydrolyzed when exposed to a mildly acidic environment; and

n is an integer greater than 10.

2. The stimuli-responsive polysaccharide antimicrobial agent of claim 1, wherein

R^!-R¹⁰ are each independently selected from H, -CH₃, -CH2CH3, -OCH3, and - OCH2CH3, an optionally substituted (Ci-Ci2)alkyl group, an optionally substituted (Ci- Ci2)heteroalkyl group, an optionally substituted (Ci-Ci2)alkenyl group, an optionally substituted (Ci-Ci2)heteroalkenyl group, an optionally substituted (Ci-Ci2)alkynyl group, an optionally substituted (Ci-Ci2)heteroalkynyl group, an optionally substituted (C4- C8)cylcoalkyl group, an optionally substituted aryl group, or an optionally substituted heterocycle, wherein R² and R³ may be connected to each other to form a ring structure, and wherein R⁷ and R⁸ may be connected to form a ring structure.

3. The stimuli-responsive polysaccharide antimicrobial agent of claim 1, wherein the stimuli-responsive polysaccharide antimicrobial agent comprises the structure of Formula 1(a):

Formula 1(a)

or a pharmaceutically acceptable salt thereof,

wherein,

R¹ is a substituted (Ci-Ci2)alkyl group, a substituted (Ci-Ci2)heteroalkyl group, a substituted (Ci-Ci2)alkenyl group, a substituted (Ci-Ci2)heteroalkenyl group, a substituted (Ci-Ci2)alkynyl group, a substituted (Ci-Ci2)heteroalkynyl group, a substituted (C₄- C8)cylcoalkyl group, a substituted aryl group, or an optionally substituted heterocycle, wherein R¹ comprises at least a substitution or group that can be protonated in an acidic environment;

R⁶ is a substituted (Ci-Ci2)alkyl group, a substituted (Ci-Ci2)heteroalkyl group, a substituted (Ci-Ci2)alkenyl group, a substituted (Ci-Ci2)heteroalkenyl group, a substituted (Ci-Ci2)alkynyl group, a substituted (Ci-Ci2)heteroalkynyl group, a substituted (C₄- C8)cylcoalkyl group, a substituted aryl group, or an optionally substituted heterocycle, wherein R⁶ comprises at least a substitution or group that can be protonated in an acidic environment;

R², R³, R⁷, and R⁸ are each independently selected from H, -CH₃, -CH2CH3, -OCH3, or -OCH2CH3, wherein R² and R³ may be connected together to form a ring structure selected be connected together to form a

ring structure selected from or and

n is an integer greater than 100

4. The stimuli-responsive polysaccharide antimicrobial agent of claim 3, wherein R¹ and R⁶ is substituted with an amino group or a sulfhydryl group.

5. The stimuli-responsive polysaccharide antimicrobial agent of claim 3, wherein the stimuli-responsive polysaccharide antimicrobial agent comprises the structure of Formula 1(b):

Formula 1(b)

or a pharmaceutically acceptable salt thereof,

wherein,

R³ may be connected together to form a ring structure selected

wherein R⁷ and R⁸ may be connected together to form

structure selected from and

n is an integer greater than 10

6. The stimuli-responsive polysaccharide antimicrobial agent of claim 5, wherein the stimuli-responsive polysaccharide antimicrobial agent comprises the structure of Formula 1(c):

Formula 1(c)

or a pharmaceutically acceptable salt thereof, wherein n is an integer greater than 10.

7. The stimuli-responsive polysaccharide antimicrobial agent of claim 1, wherein the stimuli-responsive polysaccharide antimicrobial agent exhibits the following properties: in a neutral aqueous environment, the stimuli-responsive polysaccharide antimicrobial agent is generally water soluble; and

in a mildly acidic aqueous environment, the stimuli-responsive polysaccharide antimicrobial agent is hydrolyzed to chitosan.

8. A composition comprising the stimuli-responsive polysaccharide antimicrobial agent of any one of claims 1 to 7 complexed with or polyplexed with one or more nucleic acids; antibiotics; proteins; a CRISPR-Cas system, a CRISPRi system, a CRISPR-Cpfl system; and/or antifungal agents.

9. The composition of claim 8, wherein the one or more nucleic acids are

deoxyribonucleic acid (DNA), polydeoxyribonucleotide (pdrn) and/or complementary DNA (cDNA).

10. The composition of claim 8, wherein the one or more nucleic acids are ribonucleic acids (RNAs).

11. The composition of claim 10, wherein the one or more nucleic acids are small interfering RNAs (siRNAs), CRISPR RNAs, small non-coding microRNAs (miRNAs), and/or antisense RNAs (asRNAs).

12. The composition of claim 11, wherein the siRNAs, CRISPR RNAs, miRNAs or asRNAs target genes necessary for fungal or bacterial growth or replication, target genes for antibiotic resistance, target host genes that are involved in the immune response caused by a fungal or bacterial infection, and/or target the host genes involved in mediating the entry of bacteria or fungi into host cells.

13. The composition of claim 8, wherein the stimuli-responsive polysaccharide antimicrobial agent is complexed or polyplexed with a CRISPR-Cas system, a CRISPRi system, or a CRISPR-Cpfl system that targets microbial genes responsible for antibiotic or antifungal resistance, or targets microbial genes for microbe growth and survival, or targets microbial genes that result in the death of the microbe.

14. The composition of claim 8, wherein the one or more antibiotics is selected from Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Streptomycin, Spectinomycin, Geldanamycin, Herbimycin, Rifaximin, Loracarbef,

Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin,

Cephradine, Cephapirin, Cephalothin, Cefalexin, Cefaclor, Cefoxitin, Cefotetan,

Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline, Tinidazole, and Trimethoprim.

15. The composition of claim 8, wherein the one or more antifungal agents is selected from itraconazole, posaconazole, ketoconazole, clotrimazole, miconazole, voriconazole, caspofungin, anidulafungin, micafungin, nystatin, amphotericin b, griseofulvin, terbinafine, and/or flucytosine.

16. The composition of claim 11, wherein the stimuli-responsive polysaccharide antimicrobial agent is complexed with or polyplexed with pdrn so as to form particles having diameters greater than 10 nm.

17. The composition of claim 16, wherein the particles have diameters of around 200 nm.

18. The composition of claim 16, wherein the polyplexes have a N/P ratio of about 100.

19. The composition of claim 16, wherein the particles are highly monodisperse particles with a polydispersity index of less than 0.1.

20. A pharmaceutical composition comprising the stimuli-responsive polysaccharide antimicrobial agent of any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition is formulated for oral, parenteral or topical administration.

22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition if formulated for parenteral administration and comprises up to 200 mg/kg of the stimuli-responsive polysaccharide antimicrobial agent.

23. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition further comprises one or more additional therapeutic agents.

24. The pharmaceutical composition of claim 23, wherein the one or more additional therapeutic agents are one or more antibiotics.

25. The pharmaceutical composition of claim 24, wherein the one or more antibiotics is selected from Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Streptomycin, Spectinomycin, Geldanamycin, Herbimycin, Rifaximin, Loracarbef, Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil,

Cefazolin, Cephradine, Cephapirin, Cephalothin, Cefalexin, Cefaclor, Cefoxitin, Cefotetan, Cefamandole, Cefmetazole, Cefonicid, Loracarbef, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Moxalactam, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Dalbavancin, Oritavancin, Clindamycin, Lincomycin, Daptomycin, Azithromycin, Clarithromycin, Roxithromycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Azlocillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin, Ticarcillin, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam,

Ticarcillin/clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofioxacin, Lomefloxacin, Moxifloxacin, Nadifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfioxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole,

26. The pharmaceutical composition of claim 23, wherein the one or more additional therapeutic agents are antifungal agents.

27. The pharmaceutical composition of claim 26, wherein the one or more antifungal agents is selected from itraconazole, posaconazole, ketoconazole, clotrimazole, miconazole, voriconazole, caspofungin, anidulafungin, micafungin, nystatin, amphotericin b, griseofulvin, terbinafine, and/or flucytosine.

28. A method of treating a subject suspected of having or at risk of developing a microbial infection comprising administering to the subject a therapeutically effective amount of the stimuli-responsive polysaccharide antimicrobial agent of any one of claims 1 to 7.

29. The method of claim 28, wherein the subject is suspected of having or at risk of developing a bacterial and/or a fungal infection.

30. The method of claim 29, wherein the bacterial infection is caused by a bacterium selected from the group consisting of Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile,

Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enter ocolitica, and Yersinia pseudotuberculosis.

31. The method of claim 29, the fungal infection is caused by a fungus selected from the group consisting of Absidia corymbifera, Absidia ramose, Achorion gallinae, Actinomadura spp., Ajellomyces dermatididis, Aleurisma brasiliensis, Allersheria boydii, Arthroderma spp., Aspergillus flavus, Aspergillus fumigatu, Basidiobolus spp, Blastomyces spp, Cadophora spp, Candida albicans, Cercospora apii, Chrysosporium spp, Cladosporium spp, Cladothrix asteroids, Coccidioides immitis, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Cunninghamella elegans, Dematium wernecke, Discomyces israelii, Emmonsia spp, Emmonsiella capsulate, Endomyces geotrichum, Entomophthora coronate, Epidermophyton floccosum, Filobasidiella neoformans, Fonsecaea spp., Geotrichum candidum, Glenospora khartoumensis, Gymnoascus gypseus,

Trichophyton spp, Trichosporon spp, and Zopfla rosatii.

32. The method of claim 29, further comprising administering to the subject, concurrently or sequentially, one or more antibiotics, and/or antifungal agents.

33. The method of claim 32, wherein the one or more antibiotics is selected from

Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Streptomycin, Spectinomycin, Geldanamycin, Herbimycin, Rifaximin, Loracarbef,

Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin,

Cephradine, Cephapirin, Cephalothin, Cefalexin, Cefaclor, Cefoxitin, Cefotetan, Cefamandole, Cefmetazole, Cefonicid, Loracarbef, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Moxalactam, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Dalbavancin, Oritavancin, Clindamycin, Lincomycin, Daptomycin, Azithromycin, Clarithromycin, Roxithromycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Azlocillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin, Ticarcillin, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam,

Ticarcillin/clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin,

Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nadifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin,

Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole,

34. The method of claim 32, wherein the one or more antifungal agents is selected from itraconazole, posaconazole, ketoconazole, clotrimazole, miconazole, voriconazole, caspofungin, anidulafungin, micafungin, nystatin, amphotericin b, griseofulvin, terbinafine, and/or flucytosine.

35. A method of treating a subject suspected of having or at risk of developing a microbial infection comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 8 to 19.

36. The method of claim 35, wherein the subject is suspected of having or at risk of developing a bacterial and/or a fungal infection.

37. The method of claim 36, wherein the bacterial infection is caused by a bacterium selected from the group consisting of Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile,

38. The method of claim 36, the fungal infection is caused by a fungus selected from the group consisting of Absidia corymbifera, Absidia ramose, Achorion gallinae, Actinomadura spp., Ajellomyces dermatididis , Aleurisma brasiliensis, Allersheria boydii, Arthroderma spp., Aspergillus flavus, Aspergillus fumigatu, Basidiobolus spp, Blastomyces spp, Cadophora spp, Candida albicans, Cercospora apii, Chrysosporium spp, Cladosporium spp, Cladothrix asteroids, Coccidioides immitis, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Cunninghamella elegans, Dematium wernecke, Discomyces israelii, Emmonsia spp, Emmonsiella capsulate, Endomyces geotrichum, Entomophthora coronate, Epidermophyton floccosum, Filobasidiella neoformans, Fonsecaea spp., Geotrichum candidum, Glenospora khartoumensis, Gymnoascus gypseus,

Trichophyton spp, Trichosporon spp, and Zopfla rosatii.

39. The method of claim 36, wherein the subject is suspected of having an intracellular bacterial infection.

40. The method of claim 39, wherein the intracellular bacterial infection is caused by bacterium selected from the genus Chlamydophila, Ehrlichia, Rickettsia, Chlamydia, Salmonella, Neisseria, Brucella, Mycobacterium, Nocardia, Listeria, Francisella, Legionella, or Yersinia pestis.

41. A method of treating a subject suspected of having or at risk of developing a microbial infection comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 20 to 27.

42. The method of claim 41, wherein the subject is suspected of having or at risk of developing a bacterial and/or a fungal infection.

43. The method of claim 42, wherein the bacterial infection is caused by a bacterium selected from the group consisting of Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile,

44. The method of claim 42, wherein the fungal infection is caused by a fungus selected from the group consisting of Absidia corymbifera, Absidia ramose, Achorion gallinae, Actinomadura spp., Ajellomyces dermatididis, Aleurisma brasiliensis, Allersheria boydii, Arthroderma spp., Aspergillus flavus, Aspergillus fumigatu, Basidiobolus spp, Blastomyces spp, Cadophora spp, Candida albicans, Cercospora apii, Chrysosporium spp, Cladosporium spp, Cladothrix asteroids, Coccidioides immitis, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Cunninghamella elegans, Dematium wernecke, Discomyces israelii, Emmonsia spp, Emmonsiella capsulate, Endomyces geotrichum, Entomophthora coronate, Epidermophyton floccosum, Filobasidiella neoformans, Fonsecaea spp., Geotrichum candidum, Glenospora khartoumensis ,

Trichophyton spp, Trichosporon spp, and Zopfla rosatii.

45. A method to prevent an infection by a microbial agent of a wound or a burn, comprising topically administering to the subject a therapeutically effective amount of the stimuli-responsive polysaccharide antimicrobial agent of any one of claims 1 to 7.

46. A method to prevent an infection by a microbial agent of a wound or a burn, comprising topically administering to the subject a therapeutically effective amount of the composition of any one of claims 8 to 19.

47. A method to prevent an infection by a microbial agent of a wound or a burn, comprising topically administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 20, 21, and 23 to 27.

48. A method for inhibiting the growth of a microorganism or microbe by contacting the microorganism or microbe with an inhibiting effective amount of the stimuli-responsive polysaccharide antimicrobial agent of any one of claims 1 to 7.

49. The method of claim 48, wherein the microorganism or microbe is contacted in vitro with the stimuli-responsive polysaccharide antimicrobial agent.

50. The method of claim 48, wherein the microorganism or microbe is contacted in vivo with the stimuli-responsive polysaccharide antimicrobial agent.