Cryptococcal NANO-immunotherapy: a fungal drug carrier platform

Abstract

The present invention provides a fungal drug carrier comprising an avirulent fungal pathogen cell, for example a Cryptococcus neoformans cell, linked to one or more surface-bound drug-loaded nanoparticles, and methods of making the fungal drug carrier. The invention provides methods of using the fungal drug carrier to deliver a drug to the central nervous system of a patient, wherein the fungal drug carrier is phagocytosed by an immune cell of a patient, transported across the blood-brain barrier, and vomocytosed to allow drug release from the surface-bound nanoparticle into the central nervous system of the patient. The methods involve treating a central nervous system in a patient comprising administering to the patient a pharmaceutical composition comprising a fungal drug carrier.

Description

Attorney Docket No.:049648-632156 UF Ref. T19361WO001 CRYPTOCOCCAL NANO-IMMUNOTHERAPY: A FUNGAL DRUG CARRIER PLATFORM CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 USC 119(e) of U.S. Provisional Application No.63 / 665,961, filed June 28, 2024, which is incorporated by reference in its entirety for all purposes. STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under Grant No. R35 GM125012 awarded by the National Institutes of Health. The government has certain rights in the invention. This invention was created in whole or in part by funding received from the National Institutes of Health under Training Grant Number T32GM099608; and from the National Science Foundation under Fellowship Award Numbers DGE-1650042 and DGE-2236414. BACKGROUND OF THE INVENTION

[0003] More than 1.2 billion people worldwide are affected by neurological disorders, yet only 2% of approved therapies can successfully enter the central nervous system (CNS) for therapeutic effects [1], [2],

[0044] . The neurological disorder amyotrophic lateral sclerosis (ALS) or Lou Gehrig’s disease which afflicts more than 200,000 people worldwide has very few and only slightly effective treatment options [3]. This limited efficacy can be attributed to physiological fortresses such as the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) and the physicochemical properties (solubility, molecular weight, stability, etc.) of the FDA-approved drugs for ALS [2]. The two most commonly used drugs to treat ALS (Riluzole and Edaravone) are lipophilic and <400 Da in molecular weight, key properties for small molecules to diffuse across the BBB and BSCB passively [4], [5]. However, these favorable properties also render these drugs susceptible to off-target tissue absorption upon systemic administration, thus reducing their bioavailability in the CNS [6]. Recent literature has shown that drug encapsulation with nanomaterials is a promising solution for reducing absorption in 1Attorney Docket No.:049648-632156 UF Ref. T19361WO001 off-target tissues because the nanomaterial essentially masks the drug's physicochemical properties [7]. However, these nanoscale formulations still fail to target the CNS due to the tissue’s physiological barriers, and the body’s various clearance mechanisms [8], [9]. Therefore, there is a need for a more effective drug delivery platform that can bypass and withstand these barriers to treat ALS.

[0004] The fungus Cryptococcus neoformans (Cn or CN) infiltrates the CNS and induces cryptococcal meningitis. Cn is typically inhaled and subsequently circumvents the innate immune system of the lung escaping into circulation as a free cell or while engulfed by a phagocyte

[0010] . In circulation, Cn uses three mechanisms to cross the BBB efficiently: 1) paracellular transport (for example by penetrating tight junctions) 2) receptor-mediated transcytosis (receptor-mediated transport), and 3) hitchhiking phagocytic cells (for example cell- mediated transport) and executing vomocytosis (“non-lytic exocytosis”) to interact with the CNS parenchyma

[0011] ,

[0012] ,

[0013] . During vomocytosis, phagocytes expel ingested Cn from the phagolysosome without damaging the Cn or the phagocyte itself. These normally pathogenic attributes make Cn an attractive candidate for designing a novel CNS-targeted drug delivery platform

[0014] .

[0005] There is a need in the art for improved methods for drug delivery to the central nervous system for treatment of central nervous system disorders such as ALS. BRIEF SUMMARY OF THE CLAIMED INVENTION

[0006] In one aspect, the invention provides a fungal drug carrier composition comprising an avirulent fungal pathogen cell and at least one drug-loaded nanoparticle (NP) linked to the surface of the avirulent fungal pathogen cell, wherein the avirulent fungal pathogen cell is coated with a first cationic polymer. In another aspect, the invention provides a fungal drug carrier composition comprising an avirulent fungal pathogen cell and at least one drug-loaded nanoparticle (NP) linked to the surface of the avirulent fungal pathogen cell, wherein the at least one drug-loaded nanoparticle (NP) comprises a first cationic polymer.

[0007] In some fungal drug carriers, the avirulent fungal pathogen cell is Cryptococcus or Candida albicans. In some fungal drug carriers, the avirulent fungal pathogen cell is 2Attorney Docket No.:049648-632156 UF Ref. T19361WO001 Cryptococcus neoformans , Cryptococcus gatti, or Cryptococcus grubii. In some fungal drug carriers, the avirulent fungal pathogen cell is Cryptococcus neoformans. In some fungal drug carriers, the avirulent fungal pathogen cell is Cryptococcus neoformans H99, Cryptococcus neoformans tps1Δ, or Cryptococcus neoformans plb1.

[0008] In some fungal drug carriers, the first cationic polymer is polyethyleneimine (PEI). In some fungal drug carriers, the NP is coated with a second cationic polymer. In some fungal drug carriers, the second cationic polymer is poly-L-lysine. In some fungal drug carriers, the NP comprises poly(lactic-co-glycolic) acid (PLGA) or polycaprolactone (PCL).

[0009] In some fungal drug carriers, the NP is a lipid nanoparticle, liposome, or liposomic formulation. In some fungal drug carriers, the NP comprises an inorganic material. In some fungal drug carriers, the NP is a gold (Au) NP or an iron oxide (Fe2O3)NP.

[0010] In some fungal drug carriers, the NP is spherical. In some fungal drug carriers, the NP has a diameter of about 100 nm to about 500 nm. In some fungal drug carriers, the NP has a diameter of about 100 nm to about 300 nm. In some fungal drug carriers, the NP has a diameter of about 200 nm to about 250 nm. In some fungal drug carriers, the NP has a diameter of about 200 nm. In some fungal drug carriers, the NP has a diameter of about 400 nm to about 500 nm.

[0011] In some fungal drug carriers, the NP comprises poly(lactic-co-glycolic) acid (PLGA).

[0012] In some fungal drug carriers, the NP is linked to the surface of the avirulent fungal pathogen cell by electrostatic adsorption. In some fungal drug carriers, the NP is linked to the surface of the avirulent fungal pathogen cell by conjugation.

[0013] In some fungal drug carriers, the conjugation of the NP to the avirulent fungal pathogen cell is via a surface amino group on the avirulent fungal pathogen cell. In some fungal drug carriers, the conjugation of the NP to the avirulent fungal pathogen cell is via a surface thiol group on the avirulent fungal pathogen cell.

[0014] In some fungal drug carriers, the NP is loaded with a small molecule drug or a biologic drug. In some fungal drug carriers, the NP is loaded with an anticancer drug, antibiotic drug, or analgesic drug. In some fungal drug carriers, the NP is loaded with Riluzole or Edaravone. 3Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0015] In another aspect, the invention provides a method of making a fungal drug carrier, wherein the method comprises: (a) providing an avirulent fungal pathogen cell; (b) coating the avirulent fungal pathogen cell with a first cationic polymer; (c) preparing a nanoparticle (NP) comprising a drug; and (d) linking the NP comprising the drug to the surface of the avirulent fungal pathogen coated with the first cationic polymer. Some such methods further comprise a step of coating the NP comprising the drug with a second cationic polymer after step (c). In some such methods, the linking of step(d) is by electrostatic adsorption. In some such methods, the linking of step(d) is by conjugation. Some such methods further comprise a step of functionalizing the PLGA NP comprising the drug before step(d). Some such methods further comprise a step of functionalizing the avirulent fungal pathogen cell before step (d). In some such methods, the conjugation of step (d) comprises reacting the PLGA-Azide NP comprising the drug with avirulent fungal pathogen cell functionalized by reaction with Dibenzocyclooctyne (DBCO).

[0016] In another aspect, the invention provides a method of making a fungal drug carrier, wherein the method comprises: (a) providing an avirulent fungal pathogen cell; (b) preparing a nanoparticle (NP) comprising a drug and a first cationic polymer; and (c) linking the NP comprising the drug and the first cationic polymer to the surface of the avirulent fungal pathogen. Some such methods further comprise a step of coating the NP comprising the drug and the first cationic polymer with a second cationic polymer after step (b). In some such methods, the linking of step(c) is by electrostatic adsorption. In some such methods, the linking of step(c) is by conjugation. Some such methods further comprise a step of functionalizing the PLGA NP comprising the drug before step (c). Some such methods further comprise a step of functionalizing the avirulent fungal pathogen cell before step (c). In some such methods, the conjugation of step (c) comprises reacting the PLGA-Azide NP comprising the drug with avirulent fungal pathogen cell functionalized by reaction with Dibenzocyclooctyne (DBCO).

[0017] In some methods, the avirulent fungal pathogen cell is Cryptococcus or Candida albicans. In some methods, the avirulent fungal pathogen cell is Cryptococcus neoformans , Cryptococcus gatti, or Cryptococcus grubii. In some methods, the avirulent fungal pathogen cell is Cryptococcus neoformans. In some methods, the avirulent fungal pathogen cell is 4Attorney Docket No.:049648-632156 UF Ref. T19361WO001 Cryptococcus neoformans H99, Cryptococcus neoformans tps1Δ, or Cryptococcus neoformans plb1..

[0018] In some methods, the first cationic polymer is polyethyleneimine (PEI). In some methods, the NP comprises poly(lactic-co-glycolic) acid (PLGA) or polycaprolactone (PCL).

[0019] In some methods, the NP is a lipid nanoparticle, liposome, or liposomic formulation. In some methods, the NP comprises an inorganic material. In some methods, the NP is a gold (Au) NP or an iron oxide (Fe2O3)NP.

[0020] In some methods, the NP is spherical. In some methods, the NP has a diameter of about 100 nm to about 500 nm. In some methods, the NP has a diameter of about 100 nm to about 300 nm. In some methods, the NP has a diameter of about 200 nm to about 250 nm. In some methods, the NP has a diameter of about 200 nm. In some methods, the NP has a diameter of about 400 nm to about 500 nm.

[0021] In some methods, the NP comprising the drug is a PLGA-NP.

[0022] In some methods, the second cationic polymer is poly-L-lysine.

[0023] In some methods, the conjugation is via a surface amino group on the avirulent fungal pathogen cell. In some methods, the conjugation is via a surface thiol group on the avirulent fungal pathogen cell.

[0024] In some methods, the PLGA NP comprising the drug is functionalized by reaction with EDC and sulfo-NHS to form an NHS-ester-functionalized PLGA NP comprising a drug. In some methods, the PLGA NP comprising the drug further comprises PLGA-maleimide. In some methods, the PLGA NP comprising the drug further comprises PLGA-NHS. In some methods, the PLGA NP comprising the drug further comprising PLGA-NHS is functionalized by reaction with amine-PEG-azide to form a PLGA-Azide NP comprising a drug.

[0025] In some methods, the avirulent fungal pathogen cell is functionalized by reaction with Dibenzocyclooctyne (DBCO). 5Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0026] In some methods, the drug is a small molecule drug or a biologic drug. In some methods, the drug is an anticancer drug, antibiotic drug, or analgesic drug. In some methods, the drug is Riluzole or Edaravone.

[0027] In another aspect, the invention provides a method of delivering a drug to a central nervous system of a subject, comprising the steps of: (a) providing any of the above-mentioned fungal drug carriers; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell across the blood-brain barrier or blood-spinal cord barrier into the central nervous system of the subject, and vomocytosed from the immune cell, delivering the drug to the central nervous system of the subject.

[0028] In some methods, the drug is delivered to the brain of the subject. In some methods, the drug is delivered to the spinal cord of the subject.

[0029] In another aspect, the invention provides a method of delivering a drug to a lymph node of a subject, comprising the steps of: (a) providing any of the above-mentioned fungal drug carriers; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the lymph node of the subject, and vomocytosed from the immune cell, delivering the drug to the lymph node of the subject.

[0030] In another aspect, the invention provides a method of delivering a drug to a spleen of a subject, comprising the steps of: (a) providing any of the above-mentioned fungal drug carriers; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the spleen of the subject, and vomocytosed from the immune cell, delivering the drug to the spleen of the subject.

[0031] In another aspect, the invention provides a method of delivering a drug to a pancreas of a subject, comprising the steps of: (a) providing any of the above-mentioned fungal drug carriers; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the pancreas of the 6Attorney Docket No.:049648-632156 UF Ref. T19361WO001 subject, and vomocytosed from the immune cell, delivering the drug to the pancreas of the subject.

[0032] In another aspect, the invention provides a method of delivering a drug to a lung of a subject, comprising the steps of: (a) providing any of the above-mentioned fungal drug carriers; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the lung of the subject, and vomocytosed from the immune cell, delivering the drug to the lung of the subject.

[0033] In another aspect, the invention provides a method of delivering a drug to a heart of a subject, comprising the steps of: (a) providing any of the above-mentioned fungal drug carriers; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the heart of the subject, and vomocytosed from the immune cell, delivering the drug to the heart of the subject.

[0034] In another aspect, the invention provides a method of delivering a drug to a liver of a subject, comprising the steps of: (a) providing any of the above-mentioned fungal drug carriers; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the liver of the subject, and vomocytosed from the immune cell, delivering the drug to the liver of the subject.

[0035] In another aspect, the invention provides a method of delivering a drug to a kidney of a subject, comprising the steps of: (a) providing any of the above-mentioned fungal drug carriers; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the kidney of the subject, and vomocytosed from the immune cell, delivering the drug to the kidney of the subject.

[0036] In some such methods, the administering is selected from the group consisting of intranasal, intraperitoneal, intramuscular, intravenous, and subcutaneous.

[0037] In another aspect, the invention provides a method of treating a disorder in a subject, the method comprising administering any of the above-mentioned fungal drug carriers to the subject. 7Attorney Docket No.:049648-632156 UF Ref. T19361WO001 In some such methods, the administering is selected from the group consisting of intranasal, intraperitoneal, intramuscular, intravenous, and subcutaneous.

[0038] In some such methods, the disorder is a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease. In some such methods, the central nervous system disorder is selected from the group consisting of cancer, epilepsy, stroke, infection, traumatic brain injury, spinal cord injury, a neurodegenerative disorder, amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. In some such methods, the central nervous system disorder is amyotrophic lateral sclerosis.

[0039] In some such methods, the subject is a mammal. In some such methods, the mammal is a primate. In some such methods, the mammal is a non-human primate. In some such methods, the mammal is a human. In some such methods, the mammal is a rodent. In some such methods, the rodent is a mouse, rat, guinea pig, hamster, or gerbil. In some such methods, the mammal is selected from the group consisting of human, baboon, chimpanzee, monkey, cynomolgus, marmoset, rhesus, rodent, rabbit, cat, dog, horse, cow, sheep, goat, pig, ferret, guinea pig, hamster, and gerbil. In some such methods, the subject is a non-human mammal, bird, reptile, amphibian, or fish.

[0040] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figures 1A and 1B depict fungal drug carrier platform targeting the CNS.

[0042] Figures 2A-2B are schematics depicting Cryptococcus neoformans (Cn) Infiltration of the Brain. 2A (Cryptococcus neoformans (Cn) Infiltrates the Brain via Multiple Mechanisms); 2B: Cryptococcus neoformans Crosses the BBB By Hitchhiking Immune Cells 8Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0043] Figure 3 is a schematic depicting phagocytosis and vomocytosis.

[0044] Figures 4A-4B are schematics depicting use of CN for therapeutic delivery to the brain. 4A (Comparison of standard drug delivery to CN (“Bug”)-Mediated Drug Delivery); 4B: CN- Mediated Drug Delivery.

[0045] Figure 5 depicts adsorption of PLGA NPs to Cn surface. A) Fluorescence microscopic image of a fungal carrier’s cell wall (blue), PEI coating (pink), and surface-bound NPs (turquoise). B) Flow cytometric data of NP adsorption to Cn surface. C) Flow cytometric data of NP adsorption to Cn surface after serum incubation. D) Median fluorescence intensity (MFI) of Rhod-123+CFW+population before (orange) and after serum (blue) for PEI-coated Cn+PLGA NPs (adsorbed) and PEI-coated Cn+PLGA-NHS NPs (conjugated). E) Zeta potentials (mV) of fungal carrier components.

[0046] Figure 6 depicts experiments showing viability of Cn engineered with PEI and PLGA nanoparticles.

[0047] Figure 7 is a table summarizing results of CN biodistribution experiments.

[0048] Figure 8 is a schematic depicting Layer-By-Layer Biodistribution Study

[0049] Figure 9 depicts Layer-By-Layer FDC Biodistribution

[0050] Figures 10A-10B depict tethering of NPs to CN cells.10A: Schematic of tethering of CN cells to NPs by adsorption and by conjugation. 10B: Schematic of PLGA NP Synthesis.

[0051] Figure 11A-11B depict physical properties of fungal carrier components. 11A: PLGA NP Dynamic Light Scattering: 11B: Cn (H99 Strain) micrograph.

[0052] Figures 12A-12B depict results showing NP and Cn have similar surface charges.12A: Surface Charge of Fungal Drug Carrier Components; 12B: Schematic showing similarly charged Cn and NP surfaces likely result in repulsive Coulomb forces and reduced conjugation efficiency. 9Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0053] Figure 13 is a schematic depicting Leveraging Electrostatic Interactions for NP Attachment to Cn (top panel) Layer-by-Layer Approach; (bottom panel) Single-Layered Approach

[0054] Figure 14 is a schematic depicting Layer-By-Layer Approach to FDC Synthesis.

[0055] Figure 15 depicts results of flow cytometry studies showing that LBL Approach Enables PLGA NP Adsorption to the Cn Surface.

[0056] Figures 16A-16B depict results of studies Quantifying NPs on Cn Surface.16A: PLGA NP Standard Curve; 16B: Estimate (based on fluorescence) of number of NPs attached to Cn surface (NPs / FDC). Target ratio: 200 NP-to-1 Cn ‘Ratio NP:Cn’ refers to the starting conjugation conditions

[0057] Figure 17 is a schematic depicting Serum Test to Evaluate Coupling Stability; (left panel) Schematic of PBCN Incubation with serum (right panel) schematic showing expanded view of serum protein-mediated particle desorption.

[0058] Figure 18-depicts a schematic showing An Approach to Fungal Carrier Synthesis.

[0059] Figures 19A-19E depict results showing Fungal Carriers Are Viable and Surface-Bound Particles Are Stably Attached.19A Fungal Carrier Viability; 19B Fungal Carrier Yield After Serum Incubation; 19C: PEI-Coated Cn +PLGA Particles; 19D: PEI-Coated Cn + PLGA-NHS Particles; 19E: PEI-Coated Cn+ PLGA-Azide Particles.

[0060] Figure 20 depicts results showing FDCs Can Still Execute Vomocytosis

[0061] Figure 21 depicts a schematic showing Fungal Carrier Synthesis Protocol.

[0062] Figure 22 depicts experiments showing PEI Coating and Fungal Carrier Synthesis Result in Altered Surface Charges.

[0063] Figure 23 depicts experiments showing Surface Coating Cryptococcus neoformans with PEI Enables Electrostatic Adsorption of PLGA NPs. 10Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0064] Figure 24 depicts experiments showing PLGA Nanoparticles Remain Attached via Electrostatic Adsorption After Incubation with Serum Proteins.

[0065] Figure 25 depicts experiments showing Fungal drug carriers Remain Viable After Synthesis.

[0066] Figure 26 depicts schematic showing Conjugation Schemes.

[0067] Figure 27 depicts schematic showing Nanoparticle Synthesis.

[0068] Figure 28 depicts experiments showing Physicochemical Properties of PLGA NPs.

[0069] Figure 29 depicts schematic showing EDC / NHS Fungal Carrier Synthesis Scheme.

[0070] Figure 30 depicts schematic showing EDC / NHS FDC Synthesis Protocol.

[0071] Figure 31 depicts experiments showing EDC / NHS Rxn: Particles Successfully Attached to CN.

[0072] Figure 32 depicts schematic showing Coating the NP with a Cationic Polymer May Improve NP-to-CN Coupling.

[0073] Figure 33 depicts schematic showing Fungal Carrier Synthesis Protocol with PLL.

[0074] Figure 34 depicts experiments showing PLL Increases Surface Charge of NPs.

[0075] Figures 35A-35B depict results of experiments showing PLL Coating Increases NP Coupling to the CN Surface.35A: Flow cytometry images; 35B: NP+CN Coupling (Adsorbed vs. Conjugated)

[0076] Figure 36 depicts schematic showing Potential Conjugation of PLL to NPs May Reduce the NP Surface Charge.

[0077] Figure 37 depicts experiments showing EDC / NHS Reaction Decreases PLL-Coated NP Surface Charge. 11Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0078] Figure 38 depicts experiments showing Serum Incubation Results in Significant Loss of Surface-Bound NPs.

[0079] Figure 39 depicts experiments showing Physicochemical Properties of PLGA-MAL NPs.

[0080] Figure 40 depicts schematic showing Fungal Carrier Synthesis with PLGA-MAL NPs.

[0081] Figure 41 depicts experiments showing PLGA-MAL NPs Attach to CN, but Still Dislodge from CN Surface.

[0082] Figure 42 depicts experiments showing MFI of CN+ for PLGA-MAL NPs Does not Shift Drastically.

[0083] Figure 42 depicts experiments showing Fungal Carrier Viability is Ratio-Dependent for PLGA-MAL NP.

[0084] Figure 44 depicts schematic showing conjugation of PLGA-Maleimide NP to CN.

[0085] Figure 45 depicts schematic showing conjugation of NPs and CN by “click” chemistry.

[0086] Figures 46A-46C are schematics depicting Synthesizing PLGA-PEI NPs.46A: schematic showing how PLGA-PEI NPs were prepared and linked to CN using an adsorption approach; 46B: schematic showing how PLGA-PEI-Azide NPs were prepared and linked to DBCO- functionalized CN using a conjugation approach; 46C: schematic showing how PLGA-PEI- Azide NPs were synthesized.

[0087] Figure 47A-47B depicts results showing physicochemical properties of PLGA-PEI NPs. 48A: PLGA-NP Dynamic Light Scattering; 48B: Surface Charge of Fungal Drug Carrier Components.

[0088] Figure 48 is a schematic depicting Single-Layer Approach to FDC Synthesis.

[0089] Figures 49A-49C depicts results showing FDCs Efficiently and Stably Attach to the Cn Surface Without Compromising Intrinsic Functions.49A: Results of Flow Cytometry Studies gated on single cells; 49B: Fungal drug carrier viability; 49C: Fungal drug carrier vomocytosis rate. 12Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0090] Figures 50A-50E depict results showing PLGA NP Release Profiles at Varied pH.50A: Rhod6g-loaded PLGA-PEI NP dynamic light scattering for NP prepared from 5002A, 5004A, and 5010 PLGA; 50B: Rhod-6g-loaded PLGA PEI-NP surface charge for NP prepared from 5002A, 5004A, and 5010 PLGA; 50C: release of Rhod6G from NP prepared from 5002A PLGA, from 5004A PLGA, and from 5010 PLGA at pH 5 over time; 50D: release of Rhod6G from NP prepared from 5002A PLGA, from 5004A PLGA, and from 5010 PLGA at pH 7 over time; 50E: release of Rhod6G from NP prepared from 5002A PLGA, from 5004A PLGA, and from 5010 PLGA at pH 7.4 over time.

[0091] Figure 51 shows results of studies showing UV Irradiation Induces Time-dependent Reduction in Cn Viability; UV-Treated CN Viability

[0092] Figure 52 is a schematic showing a procedure for intracellular hydrogelation.

[0093] Figure 53 depicts schematic on Investigating Fungal Drug Carrier Efficacy in ALS.

[0094] Figure 54 depicts scanning electron microscopy micrographs of an unmodified H99 cryptococcal cell and a H99 cryptococcal cell modified with PLGA nanoparticles on its surface.

[0095] Figure 55 depicts a schematic of protocol for Bioluminescent Cn Tracking via Ex Vivo Imaging in mice treated with Tofacitinib.

[0096] Figures 56A-K depict Cn Trafficking Patterns Are Altered by Delivery Route and Immunosuppression. Average radiance of tissues excised from mice inoculated parenterally with luciferase-expressing Cn. Healthy and TFB-treated BALB / C mice were infected with 5x106Cn. Tissues of interest were excised at various time points and imaged for bioluminescence via an in vivo imaging system (IVIS). The average radiance of each tissue was normalized against the corresponding tissue from saline controls. x-axis: Time (days); y-axis for all plots: Normalized Average radiance (photons / s / cm2 / sr) [Log10]. 56A: Brain; 56B: Lungs; 56C: Heart; 56D: Liver; 56E: Spleen; 56F: Pancreas; 56G: Kidneys; 56H: Left Popliteal Lymph Node; 56I: Right Popliteal Lymph Node; 56J: Left Inguinal Lymph Node; 56K: Right Inguinal Lymph Node.

[0097] Figure 57A-K depict Average radiance of tissues excised from simvastatin-treated mice inoculated intravenously with luciferase-expressing Cn. Healthy and simvastatin-treated 13Attorney Docket No.:049648-632156 UF Ref. T19361WO001 BALB / C mice were infected with 5x106Cn. Tissues of interest were excised at various time points and imaged for bioluminescence via an in vivo imaging system (IVIS). The average radiance of each tissue was normalized against the corresponding tissue from saline controls. Cn only and Cn+Simvastatin groups were compared using a multiple unpaired t-tests (*:p≤0.05). Some of the normalized radiance values are not displayed on the logarithmic scale as they were <1 after subtracting the background bioluminescence. Circle symbols (Cn only) Square symbols (CN + Simvastatin); x-axis: Time (days); y-axis: Average Radiance (photons / s / cm2 / sr); 57A: Brain; 57B: Lungs; 57C: Heart; 57D: Liver; 57E: Spleen; 57F: Pancreas; 57G: Kidneys; 57H: Left Popliteal Lymph Node (PLN); 57I: Right Popliteal Lymph Node (PLN); 57J: Left Inguinal Lymph Node (ILN); 57K: Right Inguinal Lymph Node (ILN).

[0098] Figure 58 is a schematic of MZ101-Loaded PLGA Nanoparticle Synthesis

[0099] Figure 59 depicts MZ101 NP Physicochemical Properties: (left panel) MZ101 NPs Dynamic Light Scattering; (right panel) Surface Charge of FDC Components.

[0100] Figure 60 depicts Calculating MZ101 Loading into Nanoparticles. (left panel): Fluorescent MZ101 Standard Curve; (right panel) Tabulated data

[0101] Figure 61 is a schematic of Gemcitabine-Loaded PLGA Nanoparticle (GNP) Synthesis

[0102] Figure 62 depicts GNP Physicochemical Properties. (left panel) Gemcitabine-Loaded PLGA NPs Dynamic Light Scattering; (right panel) Surface Charge of Gemcitabine-Loaded NPs.

[0103] Figure 63 depicts Calculating Gemcitabine Loading into Nanoparticles: (left panel) Gemcitabine Standard Curve; (right panel) Tabulated data. DEFINITIONS

[0104] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1sted., 1992); Oxford Dictionary of Biochemistry and Molecular 14Attorney Docket No.:049648-632156 UF Ref. T19361WO001 Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3rded., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1sted., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4thed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0105] The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0106] The term “disease” refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology.

[0107] The term “symptom” refers to a subjective evidence of a disease, as perceived by the subject. A "sign" refers to objective evidence of a disease as observed by a physician.

[0108] The term "individual" or “subject” refers to a human or a non-human animal. The non- human subject may be, for example, a non-human animal, and may be, for example, a non- human mammal, bird, reptile, amphibian, or fish.. The “mammal” may include any animal classified as such, including humans, non-human primates, primates, baboons, chimpanzees, monkeys, cynomolgus, marmoset, rhesus, rodents (e.g., mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, ferrets, guinea pigs, hamsters, gerbils etc.

[0109] Examples of a central nervous system disorder include cancer, epilepsy, stroke, infection, traumatic brain injury, spinal cord injury, and neurodegenerative disorders, for example, amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease), Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis, , a brain disorder, and a spinal cord disorder. 15Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0110] The terms "therapeutically effective dose," or "therapeutically effective amount," refer to that amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease. A therapeutically effective amount will, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, or reduce the risk of occurrence of one or more symptoms of a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease. The effective amount can be determined by methods well known in the art and as described in subsequent sections of this description.

[0111] The terms "treatment," "therapeutic method," and their cognates refer to treatment and prophylactic / preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely receptiveness to treatment of a subject having the disorder. Treatment may include slowing or reversing the progression of a disorder.

[0112] The term “vomocytosis” (also known as “non-lytic exocytosis”) refers to process of a phagocyte expelling a cryptococcal cell without causing damage to the phagocyte or the cryptococcal cell.

[0113] The term “transcytosis” refers to transport of a macromolecule across the interior of a cell, wherein a macromolecule is captured in a vesicle on one side of the cell, drawn across the cell, and ejected on the other side of the cell. The term “receptor-mediated transcytosis” refers to a process of 3 steps 1) receptor-mediated endocytosis of the molecule on one side of the cell, e.g. the luminal side; 2) movement of the molecule through the intracellular compartment 16Attorney Docket No.:049648-632156 UF Ref. T19361WO001 typically within the endosomal system; and 3) exocytosis of the molecule to the extracellular space on the other side of the cell, e.g. the abluminal side. The term “paracellular infiltration” refers to transport of a macromolecule through the extracellular space between adjacent cells. The term “phagocytosis” refers to process by which a cell uses its plasma membrane to engulf a large particle (≥ 0.5 μm), giving rise to an internal compartment called the phagosome.

[0114] The term “immune cell” refers to a white blood cell, for example a granulocyte, lymphocyte, or monocyte. The term “phagocyte” refers to a cell that can ingest a foreign particle, bacterium, or dead or dying cell. Exemplary phagocytes are white blood cells (such as neutrophils, monocytes, macrophages, mast cells, and dendritic cells). The term “monocyte” refers to a type of leukocyte or white blood cell that can differentiate into a macrophage or a monocyte-derived dendritic cell. The term “macrophage” refers to a white blood cell of the innate immune system that engulfs and digests pathogens, such as cancer cells, microbes, cellular debris, and foreign substances, which do not have proteins that are specific to healthy body cells on their surface, by phagocytosis. This process is called phagocytosis, which acts to defend the host against infection and injury. The term “neutrophil” refers to a type of white blood cell. A neutrophil is a type of phagocyte. The term “dendritic cell” refers to an antigen- presenting cell of the mammalian immune system and whose main function is to process antigen material and present it on the cell surface to the T cells of the immune system.

[0115] The term “blood-brain barrier” or “BBB” refers to a highly selective semipermeable border of endothelial cells that regulates the transfer of solutes and chemicals between the circulatory system and the central nervous system, thus protecting the brain from harmful or unwanted substances in the blood. The term “blood-spinal cord barrier” or “BSCB” refers to a semipermeable anatomical interface that consists of the specialized small blood vessels that surround the spinal cord, thus protecting the spinal cord from potentially toxic substances within the blood while still delivering necessary molecules to maintain spinal cord activities. Mechanisms of crossing the blood brain barrier include receptor-mediated transport, penetration of tight junctions, and immune cell extravasation.

[0116] The term “fungal pathogen” refers to a fungus that causes disease in humans or other organisms. The term “avirulent fungal pathogen” refers to a fungal pathogen modified such 17Attorney Docket No.:049648-632156 UF Ref. T19361WO001 that it no longer causes disease in humans or other organisms. The phrase “surface of a fungal pathogen” refers to the pathogen’s cell wall or polysaccharide capsule.

[0117] The term “Cryptococcus neoformans” refers to an encapsulated yeast belonging to the class Tremellomycetes and an obligate aerobe that can live in both plants and animals. Its teleomorph is a filamentous fungus, formerly referred to Filobasidiella neoformans. In its yeast state, it is often found in bird excrement. Cryptococcus neoformans can cause disease in apparently immunocompetent, as well as immunocompromised, hosts

[0061] .

[0118] The phrase “attached to” refers to the electrostatic adsorption of a particle to the cell surface without the formation of a chemical bond. The phrase “conjugated to” refers to the formation of a chemical bond via a chemical reaction of two functional groups. The term “functionalized” refers to the addition of a functional reactive group to facilitate downstream conjugation of two entities. The terms “linked to” or “coupled to” or “tethered to” or “complexed with” refer to particles attaching to the Cn surface and indicate that the two entities (NPs and Cns) have been made one via either physical or chemical means.

[0119] The term “nanoparticle” refers to a particle of matter of about 1 nanometer to about 500 nanometers (nm) in diameter. The term “drug-loaded nanoparticle” refers to a nanoparticle comprising a drug.

[0120] The term “’click’ chemistry” refers to the simple, highly reactive, and highly efficient conjugation of two entities via two reactive groups with a selective affinity towards each other.

[0121] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a fungal drug carrier may contain a fungal drug carrier alone or in combination with other ingredients. When the disclosure refers to a feature comprising specified elements, the disclosure should alternative be understood as referring to the feature consisting essentially of or consisting of the specified elements. Moreover, elements that are shown or described as being combined with other elements, can, in various embodiments, exist as stand-alone elements. 18Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0122] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.

[0123] Unless otherwise apparent from the context, the term “about” encompasses insubstantial variations, such as values within a standard margin of error of measurement (e.g., SEM or standard deviation) of a stated value. Unless otherwise apparent from the context, the term “about” encompasses values within ±5% or ±10% of a stated value.

[0124] Statistical significance means p≤0.05.

[0125] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a drug-loaded nanoparticle” or “at least one drug-loaded nanoparticle” can include a plurality of drug-loaded nanoparticle, including mixtures thereof. DETAILED DESCRIPTION I. General

[0126] The present invention provides fungal drug carrier (FDC) compositions. Some fungal drug carriers comprise an avirulent fungal pathogen cell, for example a Cryptococcus neoformans (also referred to as CN or Cn) cell, linked to one or more surface-bound drug-loaded nanoparticles (NP), wherein the avirulent fungal pathogen cell is coated with a first cationic polymer, for example polyethyleneimine (PEI). Some fungal drug carriers comprise an avirulent fungal pathogen cell, for example a Cryptococcus neoformans (also referred to as CN or Cn) cell, linked to one or more surface-bound drug-loaded nanoparticles (NP), wherein the one or more drug-loaded nanoparticles (NP) comprise a first cationic polymer, for example polyethyleneimine (PEI). After administration to a patient or subject, the fungal drug carrier can be phagocytosed by an immune cell of the patient or subject. Phagocytosed fungal drug carriers are transported within the body, for example across the blood-brain barrier or blood-spinal cord barrier or to a lymph node, spleen, pancreas, lung, heart, liver, or kidney, and vomocytosed to allow gradual drug release from the surface-bound particles into the central nervous system, lymph node, spleen, pancreas, lung, heart, liver, or kidney (Figure 1A-1B). 19Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0127] The invention provides methods of using a fungal drug carrier to deliver a drug to the central nervous system (brain or spinal cord) of a patient or subject. The invention provides methods of using a fungal drug carrier to deliver a drug to the lymph node, spleen, pancreas, lung, heart, liver, or kidney of a patient or subject. The fungal drug carriers can be used to treat a disorder in a patient or subject, for example to treat a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease in a patient or subject. The fungal drug carriers are useful in treating ALS, in a patient or subject.

[0128] The invention provides methods of making a fungal drug carrier. A drug-loaded nanoparticle can be linked to the surface of an avirulent fungal pathogen cell for example by electrostatic adsorption or by conjugation. An avirulent fungal pathogen cell linked to one or more drug-loaded nanoparticles can be described as an avirulent fungal pathogen cell decorated with one or more drug- loaded nanoparticles. Drug-loaded nanoparticles useful in the methods can comprise poly(lactic-co- glycolic) acid (PLGA). NP may further comprise PLGA-Maleimide and / or PLGA-NHS.

[0129] The invention provides methods of linking the NP to an avirulent fungal pathogen cell, wherein the avirulent fungal pathogen cell is coated with a first cationic polymer, for example polyethyleneimine (PEI). The invention provides methods of linking the NP to an avirulent fungal pathogen cell, wherein the NP comprises a first cationic polymer, for example polyethyleneimine (PEI). NP may be adsorbed to an avirulent fungal pathogen cell. NP may be conjugated to an avirulent fungal pathogen cell via an amino or a thiol group on the surface of an avirulent fungal pathogen cell. PLGA NP may be modified by EDC / NHS reaction to produce NHS-functionalized PLGA NP which can be conjugated to a CN cell via an amino on the CN cell surface. NP comprising PLGA and PLGA-maleimide may be conjugated to a CN cell via a thiol group on the CN cell surface. NP comprising PLGA and PLGA-NHS can be functionalized with amine-PEG-azide to produce PLGA-Azide NP. PLGA-Azide NP can be conjugated to a CN cell functionalized by Dibenzocyclooctyne (DBCO), via “click” chemistry. NP can be coated with a second cationic polymer, for example poly-L-lysine. See for example References

[0059] and

[0060] . 20Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0130] Stably loading avirulent Cn with FDA-approved, ALS-specific drugs can leverage the yeast’s neurotropic mechanisms to shuttle these drugs to the CNS and improve therapeutic efficacy. The inventors have synthesized viable fungal drug carriers (FDCs) where cryptococcal cells have been engineered to stably carry poly(lactic-co-glycolic) acid (PLGA) nanoparticles on the cell surface.

[0131] By engineering avirulent cryptococcal cells to carry ALS-specific drugs, the targeted delivery of these drugs to the CNS is improved. Improved targeted delivery of ALS-specific drugs via Cn-mediated neurotropism improves the therapeutic efficacy of modestly effective ALS drugs approved by the FDA. II. Fungal Pathogens

[0132] Several pathogens are capable of infiltrating the CNS, for example fungal pathogens Cryptococcus neoformans (Cn), Candida albicans and Aspergillus. CN causes meningitis in immunocompromised individuals, for example those with HIV, and causes 180,000 deaths annually

[0049] . Cryptococcus neoformans (Cn) normally enters the body via inhalation. Once the fungus enters the lungs, the fungus is engulfed or trapped in the lungs to prevent dissemination to other organs. In some instances (mostly in immunocompromised people), the pathogen can escape the lungs and enter the circulation as either a free cell or while engulfed by an immune cell. While in circulation, the CN cell interacts with the blood-brain barrier and the fungus facilitates its entry to the central nervous system. Cryptococcus neoformans (Cn), a fungal pathogen, is well-equipped to cross the BBB via three distinct mechanisms: 1) paracellular infiltration 2) receptor-mediated transcytosis 3) hitchhiking phagocytic cells and executing vomocytosis to interact with the CNS parenchyma

[0011] ,

[0012] ,

[0013] (Figures 2A-2B). Cryptococcus neoformans (Cn) uses macrophages and monocytes to infiltrate the brain through vomocytosis

[0048] . The Trojan Horse-like mechanism of vomocytosis or “non-lytic exocytosis” is characterized by a phagocyte expelling a cryptococcal cell without causing damage to the phagocyte or the cryptococcal cell and is an integral mechanism for Cn dissemination to the brain. Cn relies upon monocyte phagocytosis and shuttling across the BBB and that systemic depletion of monocytes significantly decreases Cn infiltration to the brain in intranasal and intravenous infection models

[0012] ,

[0015] ,

[0016] (Figure 3). In pre-clinical infection models, Cn has been shown to traffic to the brain after IV inoculation of Cn where the pulmonary innate immune system and the BBB were dysregulated

[0034] -

[0037] . Both Cn and monocytes attach to 21Attorney Docket No.:049648-632156 UF Ref. T19361WO001 adhesion molecules and receptors overexpressed during neuroinflammation. Some CN cells have a diameter of about 5 micron. (Figure 11B). Cryptococcus neoformans is also discussed in references [27-29, 32, 39-40, 43, and 56-58].

[0133] Cryptococcus gatti, and Cryptococcus grubii are also capable of inducing vomocytosis. Exemplary fungal pathogens for use in fungal drug carriers include Cryptococcus neoformans, Cryptococcus gatti, and Cryptococcus grubii. An exemplary fungal pathogen for use in fungal drug carriers is Candida albicans.

[0134] An avirulent strain of CN is useful in FDCs of the invention to prevent the onset of unwanted diseases. Some fungal drug carriers comprise H99 Cryptococcous neoformans. Some fungal drug carriers comprise Cryptococcous neoformans tps1Δ strain. The tps1Δ strain of Cn has been shown to traffic to the brain with minimal accumulation in the lungs or spleen and not cause disease after IV administration to mice

[0019] . The tps1Δ strain is useful in FDCs for neurotropism without inducing cryptococcal meningitis. The Cryptococcus neoformans plb1 strain which has a mutation in phospholipase B is useful in FDCs of the invention

[0062] . Some fungal drug carriers comprise an engineered strain of CN.

[0135] Some CN useful in FDCs of the invention are hydrogelated [65-68]. Hydrogelation of CN can make the CN non-replicating To prepare hydrogelated CN, CN are combined with polyethylene glycol diacrylate and a photoinitiator, eosin-y, for example to first make CN infused with hydrogel monomers. The CN infused with hydrogel monomers is photoactivated by visible light to crosslink the intracellular hydrogel. Hydrogelated CN can be prepared for example as in Figure 52, Example 19.

[0136] Treatment of fungal pathogens with ultraviolet light can reduce viability [69-73]. Some FDCs of the invention are prepared using fungal pathogens treated with ultraviolet light. FDCs prepared fungal pathogens treated with UV light may exhibit improved safety for use in a patient or subject.

[0137] Viability over time, vomocytosis, and surface properties can be measured for fungal pathogens, for example CN, treated with UV light. Viability over time, vomocytosis, surface 22Attorney Docket No.:049648-632156 UF Ref. T19361WO001 properties, and viability after NP attachment (linkage) can be measured for FDCs prepared from fungal pathogens, for example CN, treated with UV light.

[0138] Lysis of yeast is discussed in reference

[0033] . III. Drug Delivery Using Engineered Cells

[0139] Over the past decade, methods have been pioneered to engineer cells to mediate drug delivery, particularly on red and white blood cells

[0020] . Given their ability to traffic the body and infiltrate inflamed or diseased tissues, blood cells are candidates for drug carriers. In preclinical and clinical applications, blood cells have been engineered to carry drug-loaded, biomaterial- based particles (“backpacks”) onto the cell surface facilitating targeted drug delivery. Particle- backpacking cells leverage the advantages of encapsulating drugs for masking the physicochemical properties and leveraging the migratory function of immune cells. This particle-backpacking approach has been demonstrated in a variety of mammalian blood cells including erythrocytes, monocytes, macrophages, lymphocytes, T cells, T Regulatory cells, and natural killer cells, with promising results in targeting neurological conditions

[0021] ,

[0022] ,

[0023] . However, the translational landscape of particle-backpacking approaches is limited since the cells would need to be isolated from patients, engineered ex vivo, and re-infused into patients, which is costly

[0024] . Despite the advances that nanotechnology provided to the field of drug delivery, these engineered nanocarrier systems

[0045] are still challenged by various systemic barriers, such as the walls of the blood vessel, the clearance mechanisms of the liver and kidneys, or the acidic conditions of endosomes at the cellular level, which prevents drug-loaded nanocarriers from reaching target tissues as well. One of the most challenging tissues to target is the brain. It is largely considered impenetrable due to the blood-brain barrier which regulates entry and exit from the brain with tremendous rigor. The present invention leverages the immune cell's ability to cross the blood-brain barrier. To reach the brain, the particle backpacking CN would rely on macrophage uptake while lymph node targeting would rely on dendritic cell uptake. (Figures 4A-4B). Immune cells are also discussed in references [46-47, and 51-55].

[0140] The inventors describe the use of biocompatible, biodegradable, and FDA-approved polymers, poly(lactic-co-glycolic) acid (PLGA), and polyethyleneimine (PEI), and FDA- 23Attorney Docket No.:049648-632156 UF Ref. T19361WO001 approved ALS drugs, Riluzole and Edaravone, to synthesize fungal drug carriers. In this approach, the drug of interest is encapsulated in a much more biocompatible material to form a drug-loaded particle or nanocarrier. PLGA is a highly tunable biomaterial that has been used in various FDA-approved drug delivery applications. PLGA is utilized to formulate negatively charged drug-loaded nanoparticles that can gradually release drugs from the Cn surface. PEI is a branched positively charged material that is used to coat Cn enabling electrostatic interactions between the drug-loaded nanoparticles and the cell surface. Like cellular backpacks, the fungal drug carriers leverage the trafficking patterns of immune cells. However, the inventors’ approach eliminates the need to isolate a patient’s cells and engineer them ex vivo. Engineered microbes as living therapeutics have gained significant attention with more than a dozen clinical trials investigating microbe-based therapeutics and even more preclinical approaches in the pipeline

[0025] . Currently, microbe-based therapeutics entail engineering microbes to respond to pathological stimuli and release therapeutics via their metabolic machinery. However, most of these clinical trials surround treating gastrointestinal conditions, which leaves a realm for engineering microbes to deliver therapeutics to other tissues. IV. Amyotrophic Lateral Sclerosis (ALS)

[0141] Amyotrophic Lateral Sclerosis (ALS) is a debilitating and fatal neurodegenerative disease. More than 200,000 people are living with ALS globally with an estimated 20,000 people dying from ALS annually in the United States alone

[0026] . ALS is a rapidly progressive neurodegenerative disease characterized by the destruction of motor neurons in the brain and spinal cord, resulting in paralysis and ultimately death by the late stages of the disease. The average life expectancy for ALS patients at the time of diagnosis ranges from 24-48 months

[0027] .

[0142] There is currently no cure for ALS, which leaves only fatal prognoses for patients. While there are seven FDA-approved drugs for treating ALS, they only elicit modest effects to slow disease progression [3]. Riluzole, the first FDA-approved drug for ALS, only slows the onset of respiratory failure and extends life expectancy by a few months

[0028] . The lack of effective therapies can be attributed to the overall lack of understanding of ALS pathophysiology where several mechanisms including glutamate excitotoxicity, protein misfolding and aggregation, and oxidative stress have been cited [3]. With several concurrent pathophysiological mechanisms at play, treating ALS becomes even more of a challenge. However, for drugs that may be able to 24Attorney Docket No.:049648-632156 UF Ref. T19361WO001 act on these mechanisms, they are limited by their ability to target the central nervous system (CNS). Therapeutics geared toward ALS must pass the blood-brain barrier (BBB) and blood- spinal cord barrier (BSCB), which prevent more than 98% of small molecules from entering the CNS

[0029] . Riluzole and Edaravone are two lipophilic small molecules (>400Da) that can passively diffuse across the CNS barriers and have been postulated to target glutamate excitotoxicity and oxidative stress, respectively. However, their lipophilicity makes them more susceptible to being absorbed by off-target tissues once they enter the circulation thus negatively influencing their therapeutic efficacy. In addition to off-target absorption, in the case of Riluzole, the dysfunctional BBB that results from ALS can further reduce CNS targeting [3]. Neurodegenerative conditions such as ALS result in dysregulated BBB and BSCB

[0030] . V. Drugs Loaded into Nanoparticles

[0143] FDCs of the invention comprise drug-loaded nanoparticles. NPs may comprise a drug for delivery to a patient or subject. Some drugs are small molecule drugs or larger biologic drugs. Some drugs are anticancer drugs, antibiotics, analgesics, or biologics. Some NPs comprise drugs useful in treatment of a central nervous system disorder, for example to treat ALS, Alzheimer’s disease, or Parkinson’s disease. Some NPs comprise drugs useful in treatment of ALS, for example Riluzole and Edaravone. Some NPs comprise a drug useful in treatment of cancer, for example gemcitabine. Some NPs comprise a drug useful in treatment of a glycogen storage disease, for example MZ-101. Some NPs comprise drugs useful in treatment of disorders of the lymph node, spleen, pancreas, lung, heart, liver, or kidney. Some NPs comprise drugs useful in treatment of pancreatic cancer, type 1 diabetes, or chronic kidney disease. Some NPs comprise drugs useful in treatment of infectious and autoimmune diseases. Some NPs comprise drugs useful in treatment of a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease. Some FDCs and methods of the invention are useful in the treatment of a disorder where CD44 is highly expressed, for example in a cancer

[0074] , a cardiovascular disorder, for example atherosclerosis

[0075] , in a liver disease, for example alcohol-associated liver disease

[0076] , or in an angiogenesis-associated disorder, for example cancer, ocular diseases, or ischemia

[0077] . Smaller drugs can diffuse out of the nanoparticles. Larger drugs can be released as the nanoparticle degrades. Some NPs include a dye Rhodamine-123. Some NPs are superparamagnetic iron-oxide nanoparticles. 25Attorney Docket No.:049648-632156 UF Ref. T19361WO001 VI. Making Fungal Drug Carriers

[0144] Nanoparticles are prepared, for example, as in

[0063] or as described, e.g., in Example 2-3 and Figures 10B and 27. Some NPs comprise an imaging agent. Imaging agents can be fluorescence / luminescence based agents for example luciferin. Imaging agents can be magnetic particle imaging (MPI) agents, for example superparamagnetic iron oxide nanoparticles (SPIONs). Imaging agents useful in positron emission topography (PET) can be used, for example fluorodeoxyglucose (FDG). Imaging agents useful in magnetic resonance imaging (MRI) can be used, for example gadolinium-based agents. Imaging agents useful in X-ray and Computed Tomography (CT) can be used, for example barium sulphate. Imaging agents useful in Single-Photon Emission Computed Tomography (SPECT) can be used, for example Technetium-99m (Tc-99m). Some NPs comprise dye Rhodamine-123. Some NPs are superparamagnetic iron-oxide nanoparticles. Exemplary NPs comprise a drug of interest for delivery to a patient or subject instead of Rhodamine-123. Some NPs comprise an imaging agent for delivery to a patient or subject. Some compositions comprise an avirulent fungal pathogen cell, for example a CN cell, linked to one or more surface-bound imaging agent-loaded nanoparticles (NP). Some NPs comprise a drug of interest and an imaging agent for delivery to a patient or subject. Some compositions comprise an avirulent fungal pathogen cell, for example a CN cell, linked to one or more surface-bound drug and imaging agent-loaded nanoparticles (NP). Some nanoparticles comprise controlled release materials, for example PLGA or polycaprolactone (PCL). Some nanoparticles comprise PLGA. A source of PLGA is Corbion. Some PLGA NPs are carboxylated. Some NPs are lipid-based (e.g., lipid nanoparticles or liposomes or liposomic formulations). Some NPs are inorganic particles (e.g.., gold (Au) NPs or iron oxide (Fe2O3)NPs).

[0145] Some NPs have a diameter of about 100 nm to about 500 nm. Some NPs have a diameter of about 100 nm to about 300 nm. Some NPs have a diameter of about 400 nm to about 500 nm. Some NPs have a diameter of about 100 nm to about 200 nm. Some NPs have a diameter of about 200 nm to about 300 nm. Some NPs have a diameter of about 200 nm to about 250 nm. Some NPs have a diameter of about 300 nm to about 400 nm. Some NPs have a diameter of about 100 nm, about 200 nm, about 300 nm, about 400 nm, or about 500 nm. Some NPs have a diameter of about 200 nm. Some NPs are spherical. Some NPs have a diameter of about 200 nm 26Attorney Docket No.:049648-632156 UF Ref. T19361WO001 and are spherical. (Figure 11A). Some NPs are rods. Some NPs are spaghetti strands. Some NPs are discoid.

[0146] NPs may be prepared by a method, for example as in Figure 27, comprising: 1. mixing an organic phase comprising PLGA and Rhodamine 123 in dichloromethane as solvent with an aqueous phase comprising 90% H2O and 10%PVA to produce an oil-in-water emulsification. 2. Homogenization to form nanoparticles 3. DCM solvent evaporation overnight in 2.5% PVA solution. 4. Particles washed via centrifugation in DI H2O and lyophilized overnight, yielding solid Rhod-123+ PLGA NPs.

[0147] MZ101-Loaded PLGA nanoparticles may be prepared by a method, for example as in Figure 58, comprising: 1. Oil-in-water emulsification of organic phase (dichloromethane as solvent, PLGA (100mg) +3 mg PEI + MZ101 (0.5 mg) and aqueous phase (10% poly-vinyl alcohol) 2. Nanoparticles formed using homogenization (25k RPM for 3 minutes). 3. DCM solvent evaporation overnight in 2.5% PVA solution. 4. Particles washed via centrifugation in DI H2O and lyophilized.

[0148] Gemcitabine-Loaded PLGA nanoparticles may be prepared by a method, for example as in Figure 61, comprising: 1. 4 mg of Gemcitabine hydrochloride (0.2 mL) added dropwise to 100 mg / mL PLGA + 3 mg / mL PEI + dichloromethane solution (4mL) 2. Homogenized the first emulsion at 25kRPM for 1 minute. 3. First emulsion added dropwise to 10%V PVA solution. 27Attorney Docket No.:049648-632156 UF Ref. T19361WO001 4. Homogenized the second emulsion at 25kRPM for 3 minutes. 5. Stirred the particles in 2.5% PVA overnight to evaporate the DCM solvent.

[0149] PLGA-NPs may be prepared by a method, for example as in Figure 10B, comprising: 1. water-in-oil emulsification of an aqueous phase comprising 90% DI H2O and 10% polyvinyl alcohol and an organic phase comprising 10 mL dichloromethane and 100 mg PLGA. 2. Homogenization to produce nanoparticles, 25 RPM for 3 minutes. 3. DCM solvent evaporation overnight in 2.5% PVA solution. 4. Particles washed via centrifugation om DI H2O and lyophilized.

[0150] Some nanoparticles comprise PLGA and PLGA-maleimide. A source of PLGA- maleimide is NanosoftPolymers. In an example, PLGA-maleimide (PLGA-MAL) polymers are mixed with PLGA at 5:1 (PLGA:PLGA-MAL) ratios in dichloromethane to formulate thiol- reactive nanoparticles (PLGA-Maleimide NP).

[0151] Some nanoparticles comprise PLGA and PLGA-NHS. A source of PLGA-NHS is RuixiBio. In an example, PLGA-NHS polymers are mixed with PLGA at 3:1 (PLGA:PLGA- NHS) ratios in dichloromethane to formulate amine-reactive nanoparticles (PLGA-NHS NP).

[0152] To form PLGA-Azide nanoparticles for “click chemistry”, PLGA-NHS nanoparticles are reacted with Amine-PEG2K-Azide overnight to produce PLGA-Azide nanoparticles. The PLGA- Azide nanoparticles are subsequently reacted with cryptococcal cells functionalized with Dibenzocyclooctyne (DBCO) to conjugate the nanoparticles to the cell surface.

[0153] Some NPs are linked to an avirulent fungal pathogen cell by electrostatic adsorption (e.g., electrostatic interaction). Some NPs are linked to an avirulent fungal pathogen cell by conjugation. See, for example, Figures 10A, 13, 18, and 26.

[0154] NPs for use in the present methods can be functionalized for conjugation to an avirulent fungal pathogen cell. 28Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0155] In some methods, NP are coated with a cationic polymer before linking to an avirulent fungal pathogen cell. In some methods the cationic polymer is poly-L-lysine. In some methods, the NP are incubated with 0.1% (w / v) poly-L-lysine solution before functionalization.

[0156] Some NPs comprise a first cationic polymer, for example polyethyleneimine (PEI). Polyethyleneimine has been used to coat yeast cells [30, 31].

[0157] Some NPs comprise PEI of Mwof about 25kDa. Some NPs are PLGA-PEI nanoparticles. PLGA-PEI NPs can be prepared, for example, by combining PLGA with branched polyethylenimine as in Figure 46. Some PLGA-PEI NPs further comprise PLGA-azide, and are referred to as PLGA-PEI-Azide NPs. PLGA-PEI-Azide NPs can be prepared, for example, by combining PLGA with branched polyethylenimine and PLGA-Azide as in Figure 46C.

[0158] An exemplary method for preparing PLGA-PEI-Azide NPs (for example as in Figure 46C) comprises: 1. Oil-in-water emulsification of: organic phase (DCM): 94% PLGA-Cy5, 3% PEI, 3% PLGA-Azide; and aqueous phase: 90% DI H20, 10% Poly-vinyl alcohol 2. Homogenize to form nanoparticle (25kRPM for 3 minutes) 3. DCM solvent evaporation overnight in 2.5% PVA solution 4. Wash particles via centrifugation in DI H20 and lyophilization.

[0159] Fungal drug carriers can be prepared by a layer-by-layer approach (for example as in Figures 13-14 and 21, Example 4). In some methods, CN are combined with PEI to prepare PEI-coated CN. PLGA-NP can be linked to PEI-coated CN by electrostatic adsorption. PLGA- NHS NPs can be conjugated to PEI-coated CN as in Figure 18.

[0160] An exemplary method for preparing FDCs by a layer-by-layer approach (as in Figure 14) comprises: 29Attorney Docket No.:049648-632156 UF Ref. T19361WO001 1. Selection of a single colony of CN grown on YPD agar. 2. Inoculation of YPD growth media for 24 hr 3. Addition of 1 colony of CN to 50 μg / mL of PEI (PBS, pH 7). Vortex gently every 10 minutes for 0.5 hr. 4. Wash excess PEI with PBS via centrifugation 3x. 5. Add PEI-coated CN to NPs (PBS, pH 7). Invert every 10 minutes for 1 hr. 6. Wash off excess NPs with a sucrose density gradient via centrifugation. 7. Collect particle-backpacking fungal carrier.

[0161] An exemplary method for preparing FDCs by a layer-by-layer approach (as in Figure 21) comprises: 1. Selection of a single colony of CN grown on YPD agar. 2. Inoculation of YPD growth media for overnight proliferation. 3. Addition of 1 colony of CFW-stained CN to 0.5 mg / mL PEI (PBS, pH 7). Stir for 30 minutes. 4. Wash excess PEI with PBS via centrifugation 3x. 5. Add PEI-coated CN to 1 mg of NPs (PBS, pH 7). Stir for 1.5 hr. 6. Wash off excess NPs with a sucrose density gradient via centrifugation. 7. Collect particle-backpacking fungal carrier.

[0162] Fungal drug carriers can be prepared by a single-layered approach, for example as in Figure 48, Example 5. In some methods, NP comprising PLGA and PEI (PLGA-PEI NPs) are first prepared by combining PLGA with PEI by combining PLGA with branched PEI. PLGA- PEI NP can be linked to CN by electrostatic adsorption. 30Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0163] An exemplary method for preparing FDCs by a single-layer approach using PLGA-PEI- Azide NPs (as in Figure 48) comprises: 1. Selection of a single colony of CN grown on YPD agar. 2. Inoculation of YPD growth media for 24 hr 3. Stain CN with calcofluor white (CFW) and conjugate with Dibenzocyclooctyne (DBCO)- NHS. 4. Wash excess CFW and DBCO with PBS via centrifugation 3x. 5. Add DBCO-CN to PLGA-PEI-Azide. Invert every 10 minutes for 1 hr. 6. Wash off excess NPs with a sucrose density gradient via centrifugation. 7. Collect particle-backpacking fungal carrier.

[0164] In some methods, carboxylated PLGA-NP are functionalized by EDC / NHS reaction, for example e.g., as in Example 4, and Figures 26 and 29-30. NHS-ester-functionalized PLGA-NP are conjugated to an amino group on surface of CN. In some methods, the carboxylated PLGA NP are coated with a cationic polymer before reaction with EDC / NHS, for example e.g., as in Example 5 and Figure 33. In some methods, the polymer is poly-L-lysine. In some methods, the carboxylated PLGA NP are mixed with 0.1% (w / v) poly-L-lysine.

[0165] Exemplary carboxylated PLGA NPs are conjugated to CN by a method comprising: 1. Carboxylated PLGA NPs particles are incubated and rotated with EDC and sulfo- NHS for 30 minutes at room temperature, producing NHS-ester-functionalized PLGA NP. 2. NHS-ester-functionalized PLGA NP are incubated with CN at room temperature for 30 minutes at room temperature to produce NHS-ester-functionalized PLGA NP conjugated to CN, and washed 3x with 0.05% Tween-20 (3k rpm for 5 minutes). 31Attorney Docket No.:049648-632156 UF Ref. T19361WO001 4. The Tween-20 washed NHS-ester-functionalized PLGA NP conjugated to CN are washed with sucrose density gradient (650 rpm for 10 minutes).

[0166] An exemplary method for conjugating PLL-coated carboxylated PLGA NPs to CN comprises: 1. Carboxylated PLGA NPs particles are incubated and rotated with 0.1% poly-L- lysine MES solution for 30 minutes at room temperature. 2. PLL polymer coated Carboxylated PLGA NPs are washed twice with MES buffer (7400xg for 15 minutes). 3. Washed PLL polymer coated Carboxylated PLGA NPs are incubated and rotated with EDC and sulfo-NHS in MES buffer for 30 minutes at room temperature, producing PLL-coated NHS-ester-functionalized PLGA NP. 4. PLL-coated NHS-ester-functionalized PLGA NP are incubated with CN at room temperature for 1.5 hr and washed 3x with 0.05% Tween-20 (1010xg for 5 minutes) to produce PLL-coated NHS-ester-functionalized PLGA NPs conjugated to CN. 4. The Tween-20 washed PLL-coated NHS-ester-functionalized PLGA NPs conjugated to CN are washed with sucrose density gradient (670 rpm for 10 minutes).

[0167] In some methods, PLGA-Maleimide NP are functionalized by thiol-maleimide reaction, for example e.g., as in Example 6 and Figures 26, 40, and 44. PLGA-maleimide NP are conjugated to a thiol group on surface of CN. In some methods, the PLGA-Maleimide NP are coated with a polymer before conjugation to a thiol group on surface of CN. In some methods, the polymer is poly-L-lysine. In some methods, the PLGA-maleimide NP are mixed with 0.1% (w / v) poly-L-lysine.

[0168] Exemplary PLGA-Maleimide NPs are conjugated to CN by a method comprising: 1. PLGA-MAL NP particles are incubated and rotated with 0.1% poly-L-lysine PBS solution for 30 minutes at room temperature. 32Attorney Docket No.:049648-632156 UF Ref. T19361WO001 2. PLL polymer coated Maleimide-functionalized NPs are washed twice with PBS (7400xg for 5 minutes). 3. Washed PLL polymer coated Maleimide-functionalized NPs are incubated with CN at room temperature for 1.5 hr and washed 3x with 0.05% Tween-20 (1010xg for 5 minutes) to produce PLGA-Maleimide NPs conjugated to CN. 4. The Tween-20 washed PLGA-Maleimide NPs conjugated to CN are washed with sucrose density gradient (670 rpm for 10 minutes).

[0169] Some NPs and CN are conjugated by “click” chemistry e.g., as in Examples 3 and 9 and Figures 26 and 45-46. In some methods, (1) PLGA-NHS NPs are functionalized by reaction with amine-PEG-azide, forming PLGA-Azide nanoparticles, (2) an amino group on the surface of CN is functionalized by reaction with Dibenzocyclooctyne (DBCO) -NCS, forming cryptococcal cells functionalized with Dibenzocyclooctyne (DBCO), and (3) the PLGA-Azide nanoparticles and cryptococcal cells functionalized with Dibenzocyclooctyne (DBCO) are conjugated by “click” chemistry

[0064] . In some methods, PLGA-NHS nanoparticles are reacted with Amine-PEG2K-Azide overnight to form PLGA-Azide nanoparticles, which are subsequently reacted with cryptococcal cells functionalized with Dibenzocyclooctyne (DBCO) to conjugate the nanoparticles to the cell surface.”

[0170] In some methods, the PLGA-NHS NP are coated with a cationic polymer before reaction with amine-PEG-azide. In some methods, the cationic polymer is poly-L-lysine. In some methods, the PLGA-NHS NP are mixed with 0.1% (w / v) poly-L-lysine.

[0171] In some methods of making fungal drug carriers, the avirulent fungal pathogen cell is coated with a first cationic polymer before linking to NP. Some avirulent fungal pathogen cells are coated with polyethyleneimine (PEI) e.g., as in Example 8 and Figures 13-14, 18, 21, and 46. In some methods of making fungal drug carriers, (1) PLGA and branched PEI are combined to make PLGA-PEI NPs; and (2) the PLGA-PEI NPs are linked to CN by adsorption, for example as in Figure 10A. In some methods of making fungal drug carriers, (1) PLGA, branched PEI, and PLGA-Azide are combined to make PLGA-PEI-Azide NPs; and (2) PLGA-PEI-Azide NPs are conjugated to DBCO-functionalized CN, for example as in Figure 10A. 33Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0172] Exemplary nanoparticles remain stably adsorbed to Cn after serum incubation (Figures 5, 19, 24, and 38 and Example 10). Fungal drug carriers will encounter the acidic environment of the phagolysosome if engulfed by a monocyte. The charge density that facilitates PEI’s stable interactions with anionic surfaces has been shown to desorb from surfaces after a pH reduction

[0031] . Cn can modulate the pH of the phagolysosome towards a less acidic pH to promote survival and potentially vomocytosis

[0032] . In some methods to promote a more stable attachment of NP to CN, PEI is conjugated or adsorbed to the Cn surface and subsequently the PLGA NPs are conjugated or adsorbed to the surface-bound PEI.

[0173] Some NPs comprise PLGA or PCL. Some NPs comprise a positively charged polymer (or a blend) as a carrier material and do not comprise a cationic polymer coating. VII. Characteristics of FDCs

[0174] Fungal drug carriers are assessed in vitro for their ability to be phagocytosed and vomocytosed by monocytes and their ability to traverse endothelial barriers (Figures 20 and 49C), optionally in comparison to wild-type CN. In addition, recent literature has cited the role that biomolecular corona formation plays on drug delivery system efficacy and biodistribution [9]. The biomolecular corona that encapsulates FDCs is sequenced to further characterize the drug delivery platform, optionally in comparison to biomolecular corona of wild-type CN.

[0175] FDCs can be assessed for in vivo biodistribution via bioluminescence imaging, for example as in Figure 8 and Example 13.

[0176] An exemplary protocol for in vivo biodistribution via bioluminescence imaging comprises: 1. Subcutaneous inoculation or intravenous inoculation of a mouse with NE1269 (luciferase-expressing CN) at dosage of 5 x 106CN / mouse.2. Intraperitoneal administration of luciferin to mouse at dosage of 150 mg / kg body weight. 3. Bioluminescent imaging to assess in vivo biodistribution, for example using Perkin Elmer IVIS CT In Vivo Imaging System (Waltham, MA). 34Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0177] An exemplary protocol for in vivo biodistribution via bioluminescence imaging , for example as in Figure 8, comprises: 1. Combining PLGA-NPs and bioluminescent CN to prepare bioluminescent fungal drug carrier (FDC) 2. Intravenous delivery of FDCs to mouse 3. Tissue excision at Days 0, 1, and 3 and collection of brain, liver, spleen, and kidneys 4. Bioluminescent imaging to assess in vivo biodistribution, for example using Perkin Elmer IVIS CT In Vivo Imaging System (Waltham, MA). 5. FDCs can be assessed for CN and NP co-localization and CN-mediated NP accumulation in the brain, for example by In Vivo Imaging Systems (IVIS) (CN tracking) and by Magnetic particle imaging (MPI) (NP tracking). VIII. Delivering Drugs Using A Fungal Drug Carrier

[0178] Upon administration to a patient, fungal drug carrier of the invention can be engulfed by an immune cell, for example a macrophage, monocyte, dendritic cell, or neutrophil. The immune cell can circulate in the body of the patient or subject, for example to the central nervous system, lymph node, spleen, pancreas, lung, heart, liver, or kidney, and vomocytose the fungal drug carrier into the central nervous system, lymph node, spleen, pancreas, lung, heart, liver, or kidney. The drug can be released from the NP in the fungal drug carrier and act on targets in the central nervous system, lymph node, spleen, pancreas, lung, heart, liver, or kidney, treating a disorder.

[0179] In some methods, the immune cell, for example a macrophage, can cross the blood-brain barrier and upon entry into the central nervous system, vomocytose the fungal drug carrier. In some methods, the immune cell can cross the blood-spinal cord barrier and upon entry into the central nervous system, vomocytose the fungal drug carrier. The drug can be released from the NP in the fungal drug carrier and act on targets in the central nervous system (e.g., brain or spinal cord), treating the central nervous system disorder. In some methods, the immune cell, for 35Attorney Docket No.:049648-632156 UF Ref. T19361WO001 example a dendritic cell, can enter a lymph node of a subject and vomocytose the fungal drug carrier into the lymph node of the subject. In some methods, the immune cell can enter a spleen of a subject and vomocytose the fungal drug carrier into the spleen. In some methods, the immune cell can enter a pancreas of a subject and vomocytose the fungal drug carrier into the pancreas of the subject. In some methods, the immune cell can enter a lung of a subject and vomocytose the fungal drug carrier into the lung of the subject. In some methods, the immune cell can enter a heart of a subject and vomocytose the fungal drug carrier into the heart of the subject. In some methods, the immune cell can enter a liver of a subject and vomocytose the fungal drug carrier into the liver of the subject. In some methods, the immune cell can enter a kidney of a subject and vomocytose the fungal drug carrier into the kidney of the subject. IX. Pharmaceutical Compositions and Methods of Use

[0180] FDCs and methods of the invention are useful in the treatment of a disorder, for example a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease. Exemplary central nervous system disorders are cancer, epilepsy, stroke, infection, traumatic brain injury, spinal cord injury, and neurodegenerative disorders, for example, amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease), Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. FDCs of the invention are useful in the treatment of pancreatic cancer, type 1 diabetes, chronic kidney disease, infectious diseases, and autoimmune diseases. Exemplary cancers are testicular cancer, breast cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, and bladder cancer. An exemplary glycogen storage disease is Pompe disease (glycogen storage disease type II). Some FDCs and methods of the invention are useful in the treatment of a disorder where CD44 is highly expressed, for example in a cancer

[0074] , a cardiovascular disorder, for example atherosclerosis

[0075] , in a liver disease, for example alcohol-associated liver disease

[0076] , or in an angiogenesis-associated disorder, for example cancer, ocular diseases, or ischemia

[0077] . Patients amenable to treatment include individuals at risk of a disorder, for example a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney 36Attorney Docket No.:049648-632156 UF Ref. T19361WO001 disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease, but not showing symptoms, as well as patients presently showing symptoms. Optionally, presence or absence of symptoms, signs, or risk factors of a disease is determined before beginning treatment.

[0181] In prophylactic applications, a fungal drug carrier or a pharmaceutical composition comprising the same is administered to a patient susceptible to, or otherwise at risk of, a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease in regime (dose, frequency, and route of administration) effective to reduce the risk, lessen the severity, or delay the onset of at least one sign or symptom of the disorder. In therapeutic applications, a fungal drug carrier is administered to a patient suspected of, or already suffering from, a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease in a regime (dose, frequency, and route of administration) effective to ameliorate or at least inhibit further deterioration of at least one sign or symptom of the disorder. Exemplary fungal drug carriers comprise a drug useful for treating a central nervous system disorder, for example Riluzole and Edaravone for treatment of ALS. Exemplary fungal drug carriers comprise a drug useful in treatment of cancer, for example gemcitabine. Exemplary fungal drug carriers comprise a drug useful in treatment of a glycogen storage disease, for example MZ-101.

[0182] A regime is considered therapeutically or prophylactically effective if an individual treated patient achieves an outcome more favorable than the mean outcome in a control population of comparable patients not treated by methods of the invention, or if a more favorable outcome is demonstrated in treated patients versus control patients in a controlled clinical trial (e.g., a phase II, phase II / III or phase III trial) at the p < 0.05 or 0.01 or even 0.001 level. 37Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0183] Effective doses vary depending on many different factors, such as means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.

[0184] The dosage depends on the condition of the patient and response to prior treatment, if any, whether the treatment is prophylactic or therapeutic and whether the disorder is acute or chronic, among other factors.

[0185] A fungal drug carrier can be administered in such doses daily, on alternative days, weekly, fortnightly, monthly, quarterly, or according to any other schedule determined by empirical analysis.

[0186] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries. The formulation depends on the route of administration chosen.

[0187] An effective amount of an FDC or a pharmaceutical composition comprising the same is an amount that is sufficient to generate a desired response, such as to reduce or eliminate a sign or symptom of a condition or disease. In some embodiments, an "effective amount" is one that treats (including prophylaxis) one or more symptoms and / or underlying causes of any central nervous system disorder, lymph node disorder, spleen disorder, pancreatic disorder, lung disorder, heart disorder, liver disorder, kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, infectious disease, autoimmune disease, cardiovascular disorder, angiogenesis-associated disorder, cancer, or glycogen storage disease. In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount that prevents one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease. 38Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0188] The pharmaceutical compositions of the invention can be readily employed in a variety of therapeutic or prophylactic applications, e.g., for treating a central nervous system disorder. In various embodiments, the pharmaceutical compositions can be used for treating or preventing a central nervous system disorder. Depending on the specific subject and conditions, pharmaceutical compositions of the invention can be administered to subjects by a variety of administration modes known to the person of ordinary skill in the art, for example, topical, intravenous, oral, subcutaneous, intraarterial, intra-articular, intracranial, intrathecal, intraperitoneal, intranasal, intraocular, parenteral, or intramuscular routes. A subcutaneous or intramuscular injection is most typically performed in the arm or leg muscles as well as the abdomen.

[0189] For prophylactic applications, the pharmaceutical composition is provided in advance of any symptom. The pharmaceutical compositions can be administered prophylactically to individuals who have a known genetic risk of a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease. Such individuals include those having relatives who have experienced such a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease, and those whose risk is determined by analysis of genetic or biochemical markers (e.g., mutations associated with a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease or a central nervous system disorder, for example mutations associated with ALS).

[0190] For therapeutic applications, the pharmaceutical composition is provided at or after the onset of a symptom of disease, for example after development of a symptom of a lymph node 39Attorney Docket No.:049648-632156 UF Ref. T19361WO001 disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease or a central nervous system disorder (e.g., ALS), or after diagnosis of the central nervous system disorder, lymph node disorder, spleen disorder, pancreatic disorder, lung disorder, heart disorder, liver disorder, kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, infectious disease, autoimmune disease, cardiovascular disorder, angiogenesis-associated disorder, cancer, or glycogen storage disease. The pharmaceutical composition of the invention can be combined with other agents known in the art for treating or preventing a central nervous system disorder (e.g., ALS) or a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease.

[0191] The subject of application of the present invention may be human or non-human. The non-human subject may be, for example, a non-human animal, and may be, for example, a non- human mammal, bird, reptile, amphibian, or fish. Examples of the non-human mammal include rodents (for example, mice and rats), dogs, cats, horses, pigs, cows, sheep, goats, primates, and the like. In addition, all aforementioned embodiments are applicable to domesticated, agricultural, or zoo-maintained mammals experiencing a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease, as well as to humans. For example, a fungal drug carrier may be administered to humans, non-human primates, primates, baboons, chimpanzees, monkeys, cynomolgus, marmoset, rhesus, rodents (e.g., mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, ferrets, guinea pigs, hamsters, gerbils etc. A fungal drug carrier may be administered to house pets such as dogs, cats, rabbits, ferrets, guinea pigs, hamsters and gerbils, as well as to agricultural animals, such as horses, sheep, cows, and pigs, or 40Attorney Docket No.:049648-632156 UF Ref. T19361WO001 to animals such as camel, cynomolgus, marmoset, rhesus and chimpanzee. A fungal drug carrier may be administered to a human.

[0192] FDCs can be administered to an animal model of disease, for example to an animal model of ALS. The biodistribution and therapeutic efficacy of FDCs is evaluated in a murine model of ALS, for example SOD1-G93A murine model of ALS. Healthy and ALS murine models are administered with fluorescently labeled FDCs loaded with superparamagnetic iron-oxide nanoparticles. FDC biodistribution can be measured via In Vivo Imaging Systems and magnetic particle imaging computed tomography tracking cryptococcal cells and PLGA nanoparticles.

[0193] Healthy and ALS murine models are treated with FDCs loaded with Riluzole and Edaravone and the disease score, grip strength and persistence on a rotating rod are measured (Figure 53).

[0194] FDCs are useful in development of drugs for treatment of disease, for example a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease.

[0195] The dosage and the frequency of administration of the pharmaceutical composition useful in methods of the present invention may be determined appropriately by a person having ordinary skill in the art (for example, a doctor) according to a target pathological condition.

[0196] Compositions and methods of the invention are useful in diagnosis of a disease or disorder in a patient or subject, for example in diagnosis of a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease, or are useful in in vivo visualization of structures, tissues, organs, systems, or organisms in a patient or subject. Compositions comprising an avirulent fungal pathogen cell, for example a CN cell, linked to one or more surface-bound imaging agent-loaded nanoparticles (NP) or linked to one or more surface-bound 41Attorney Docket No.:049648-632156 UF Ref. T19361WO001 drug and imaging agent-loaded nanoparticles (NP) are useful in in vivo visualization of structures, tissues, organs, systems, or organisms in a patient or subject. Compositions comprising an avirulent fungal pathogen cell, for example a CN cell, linked to one or more surface-bound imaging agent-loaded nanoparticles (NP) or linked to one or more surface-bound drug and imaging agent-loaded nanoparticles (NP) are useful in diagnosis of a disease or disorder in a patient or subject, for example in diagnosis of a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease in a patient or subject. Compositions comprising an avirulent fungal pathogen cell, for example a CN cell, linked to one or more surface-bound imaging agent-loaded nanoparticles (NP) or linked to one or more surface-bound drug and imaging agent-loaded nanoparticles (NP) are useful as theranostics in diagnosis and treatment of a disease or disorder in a patient or subject, for example in diagnosis and treatment of a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease in a patient or subject.

[0197] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present 42Attorney Docket No.:049648-632156 UF Ref. T19361WO001 invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. EXAMPLES

[0198] Example 1: Synthesis of fungal drug carriers.

[0199] A single colony of H99 Cn was grown overnight in yeast peptone dextrose media at 150 rpm and 30°C. The colony was subsequently washed with PBS and dispersed in a 0.5mg / mL branched polyethyleneimine (PEI) solution under stirring for 30 minutes. PEI is a cationic polymer previously used to adsorb to the negatively charged surfaces of other yeasts such as S. cerevisiae

[0033] ,

[0034] . PEI-coated Cn cells were dispersed in a PBS solution of negatively charged, NHS-terminated PLGA (PLGA-NHS) nanoparticles at 1 mg / mL and stirred for 1.5 h. Unmodified and PEI-coated cryptococcal cells were dispersed into a PBS solution of negatively charged, acid-terminated, PLGA nanoparticles to serve as negative and adsorption controls, respectively. The cryptococcal cell wall was pre-labeled with calcofluor white (CFW), and nanoparticles were pre-labeled with rhodamine-123 (Rhod-123) to distinguish the Cn and the nanoparticles. The fungal drug carriers were recovered and analyzed with flow cytometry and microscopy.

[0200] Example 2.: Synthesis of Nanoparticles

[0201] NPs were prepared by a method (Figure 27) comprising: 1. mixing an organic phase comprising PLGA and Rhodamine 123 in dichloromethane as solvent with an aqueous phase comprising 90% H2O and 10%PVA to produce an oil-in-water emulsification. 2. Homogenization to form nanoparticles 3. DCM solvent evaporation overnight in 2.5% PVA solution. 43Attorney Docket No.:049648-632156 UF Ref. T19361WO001 4. Particles washed via centrifugation in DI H2O and lyophilized overnight, yielding solid Rhod-123+ PLGA NPs. PLGA-NPs were prepared by a method (Figure 10B) comprising: 1. water-in-oil emulsification of an aqueous phase comprising 90% DI H2O and 10% polyvinyl alcohol and an organic phase comprising 10 mL dichloromethane and 100 mg PLGA. 2. Homogenization to produce nanoparticles, 25 RPM for 3 minutes. 3. DCM solvent evaporation overnight in 2.5% PVA solution. 4. Particles washed via centrifugation om DI H2O and lyophilized. PLGA-PEI-Azide NPs were prepared by a method (Figure 46C) comprising: 1. Oil-in-water emulsification of: organic phase (DCM): 94% PLGA-Cy5, 3% PEI, 3% PLGA-Azide; and aqueous phase: 90% DI H20, 10% Poly-vinyl alcohol 2. Homogenize to form nanoparticle (25kRPM for 3 minutes) 3. DCM solvent evaporation overnight in 2.5% PVA solution 4. Wash particles via centrifugation in DI H20 and lyophilization.

[0202] Some nanoparticles comprise PLGA and PLGA-maleimide. In an example, PLGA- maleimide (PLGA-MAL) polymers were mixed with PLGA at 5:1 (PLGA:PLGA-MAL) ratios in dichloromethane to formulate thiol-reactive nanoparticles (PLGA-Maleimide NP).

[0203] Example 3: Preparation of NP Comprising PLGA and PLGA-NHS 44Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0204] Some nanoparticles comprise PLGA and PLGA-NHS. In an example, PLGA-NHS polymers are mixed with PLGA at 3:1 (PLGA:PLGA-NHS) ratios in dichloromethane to formulate amine-reactive nanoparticles (PLGA-NHS NP).

[0205] To form PLGA-Azide nanoparticles for “click chemistry”, PLGA-NHS nanoparticles are reacted with Amine-PEG2K-Azide overnight to produce PLGA-Azide nanoparticles. The PLGA- Azide nanoparticles are subsequently reacted with cryptococcal cells functionalized with Dibenzocyclooctyne (DBCO) to conjugate the nanoparticles to the cell surface.

[0206] Example 4: Preparation of FDCs by a layer-by-layer approach

[0207] FDCs were prepared by layer-by-layer approach (as in Figure 14) comprising steps of: 1. Selection of a single colony of CN grown on YPD agar. 2. Inoculation of YPD growth media for 24 hr 3. Addition of 1 colony of CN to 50 μg / mL of PEI (PBS, pH 7). Vortex gently every 10 minutes for 0.5 hr. 4. Wash excess PEI with PBS via centrifugation 3x. 5. Add PEI-coated CN to NPs (PBS, pH 7). Invert every 10 minutes for 1 hr. 6. Wash off excess NPs with a sucrose density gradient via centrifugation. 7. Collect particle-backpacking fungal carrier.

[0208] Example 5: Preparation of FDCs by a single-layer approach using PLGA-PEI- Azide NPs

[0209] FDCs were prepared by single-layer approach using PLGA-PEI-Azide NPs (as in Figure 48) comprising: 1. Selection of a single colony of CN grown on YPD agar. 2. Inoculation of YPD growth media for 24 hr 45Attorney Docket No.:049648-632156 UF Ref. T19361WO001 3. Stain CN with calcofluor white (CFW) and conjugate with Dibenzocyclooctyne (DBCO)- NHS. 4. Wash excess CFW and DBCO with PBS via centrifugation 3x. 5. Add DBCO-CN to PLGA-PEI-Azide. Invert every 10 minutes for 1 hr. 6. Wash off excess NPs with a sucrose density gradient via centrifugation. 7. Collect particle-backpacking fungal carrier.

[0210] Example 6: EDC / NHS FDC Synthesis Protocol

[0211] Carboxylated PLGA NPs were conjugated to CN by a method (Figure 30) comprising: 1. Carboxylated PLGA NPs particles were incubated and rotated with EDC and sulfo-NHS for 30 minutes at room temperature, producing NHS-ester-functionalized PLGA NP. 2. NHS-ester-functionalized PLGA NP were incubated with CN at room temperature for 30 minutes at room temperature to produce NHS-ester-functionalized PLGA NP conjugated to CN, and washed 3x with 0.05% Tween-20 (3k rpm for 5 minutes). 4. The Tween-20 washed NHS-ester-functionalized PLGA NP conjugated to CN were washed with sucrose density gradient (650 rpm for 10 minutes).

[0212] Example 7: EDC / NHS FDC Synthesis Protocol with PLL

[0213] PLL-coated carboxylated PLGA NPs were conjugated to CN by a method (Figure 33) comprising: 1. Carboxylated PLGA NPs particles were incubated and rotated with 0.1% poly-L- lysine MES solution for 30 minutes at room temperature. 46Attorney Docket No.:049648-632156 UF Ref. T19361WO001 2. PLL polymer coated Carboxylated PLGA NPs were washed twice with MES buffer (7400xg for 15 minutes). 3. Washed PLL polymer coated Carboxylated PLGA NPs were incubated and rotated with EDC and sulfo-NHS in MES buffer for 30 minutes at room temperature, producing PLL-coated NHS-ester-functionalized PLGA NP. 4. PLL-coated NHS-ester-functionalized PLGA NP were incubated with CN at room temperature for 1.5 hr and washed 3x with 0.05% Tween-20 (1010xg for 5 minutes) to produce PLL-coated NHS-ester-functionalized PLGA NPs conjugated to CN. 4. The Tween-20 washed PLL-coated NHS-ester-functionalized PLGA NPs conjugated to CN were washed with sucrose density gradient (670 rpm for 10 minutes).

[0214] Example 8 : Fungal Carrier Synthesis with PLGA-MAL NPs

[0215] PLGA-Maleimide NPs were conjugated to CN by a method (Figure 40) comprising: 1. PLGA-MAL NP particles were incubated and rotated with 0.1% poly-L-lysine PBS solution for 30 minutes at room temperature. 2. PLL polymer coated Maleimide-functionalized NPs were washed twice with PBS (7400xg for 5 minutes). 3. Washed PLL polymer coated Maleimide-functionalized NPs were incubated with CN at room temperature for 1.5 hr and washed 3x with 0.05% Tween-20 (1010xg for 5 minutes) to produce PLGA-Maleimide NPs conjugated to CN. 4. The Tween-20 washed PLGA-Maleimide NPs conjugated to CN were washed with sucrose density gradient (670 rpm for 10 minutes).

[0216] Example 9: Fungal Carrier Synthesis Protocol (PLGA-NPs with where CN coated with PEI)

[0217] Fungal drug carriers were synthesized (Figure 21) by a method comprising: 1. Selection of a single colony of Cn grown on YPD agar. 47Attorney Docket No.:049648-632156 UF Ref. T19361WO001 2. Inoculation of YPD growth media for overnight proliferation. 3. Addition of 1 colony of CFW-stained Cn to 0.5 mg / mL of PEI (PBS, pH~7). Stir for 30 minutes. 4. Wash excess PEI with PBS via centrifugation 3x. 5. Addition PEI-coated Cn to 1 mg of NPs (PBS, pH~7). Stir for 1.5 h. 6. Wash off excess NPs with a sucrose density gradient via centrifugation.

[0218] Example 10: “Click” Chemistry Synthesis Protocol

[0219] Some NPs and CN are conjugated by “click” chemistry

[0064] . In some methods, (1) PLGA-NHS NPs are functionalized by reaction with amine-PEG-azide, forming PLGA-Azide nanoparticles, (2) an amino group on the surface of CN is functionalized by reaction with Dibenzocyclooctyne (DBCO) -NCS, forming cryptococcal cells functionalized with Dibenzocyclooctyne (DBCO), and (3) the PLGA-Azide nanoparticles and cryptococcal cells functionalized with Dibenzocyclooctyne (DBCO) are conjugated by “click” chemistry, for example e.g., as in Figures 26 and 45. In some methods, PLGA-NHS nanoparticles are reacted with Amine-PEG2K-Azide overnight to form PLGA-Azide nanoparticles, which are subsequently reacted with cryptococcal cells functionalized with Dibenzocyclooctyne (DBCO) to conjugate the nanoparticles to the cell surface.

[0220] Example 11: PLGA nanoparticles stably attach to the cryptococcal surface.

[0221] The negatively charged nanoparticles were shown to adsorb to the PEI-coated Cn surface as indicated by the double-positive (Rhod-123+CFW+) events (Figure 5B). Unmodified cells incubated with PLGA nanoparticles exhibited minimal double positivity, supporting the need for oppositely charged cells and nanoparticles. To assess the stability of nanoparticle-Cn attachment, each sample was dispersed into human AB serum and incubated with end-to-end rotation for 1hr. The percentage of Rhod-123+CFW+double-positive events was evaluated pre- and post-serum incubation. The median fluorescence intensities (MFI) of the Rhod-123+positive populations were used to measure if serum proteins dislodged nanoparticles since the MFI is proportional to 48Attorney Docket No.:049648-632156 UF Ref. T19361WO001 the number of nanoparticles (Figure 5C and 5D). The percentages of double-positive events and the MFIs of each sample were not drastically altered by serum incubation in both the adsorption control (PEI-Cn+PLGA) and the conjugation control (PEI-Cn+PLGA-NHS), which suggests that the electrostatic adsorption of NPs to the Cn surface could withstand opsonization in vivo. The surface charge / zeta potential of each component of the fungal drug carriers was measured to confirm the successful functionalization of cryptococcal cells (Figure 5E).

[0222] Example 12: Cryptococcal cells remain viable after fungal carrier synthesis.

[0223] Cn modified with surface-bound PEI and nanoparticles were stained with a propidium iodide viability dye and analyzed via flow cytometry. A non-viable / dead control was included to threshold viable vs. non-viable cells. The flow cytometric data indicates that the fungal carrier synthesis protocol does not drastically impact Cn viability (Figure 6).

[0224] The method comprised: 1. Inoculate media with a colony of CN overnight (30°C, 150 rpm). 2. Dose CN with UV-C light (254 nm) for 2, 4, 6, and 8 days. 3. Incubate irradiated CN at 37 °C. 4. Measure CN viability via propidium iodide assay at Day 0, Day 2, Day 4, Day 6, and Day 8.

[0225] The data demonstrate that cryptococcal cells can withstand electrostatic adsorption of nanoparticles to their surfaces.

[0226] Example 13: Cn Rapidly Traffics to the CNS After Intravenous Inoculation in Healthy Mice.

[0227] Healthy BALB / C mice were inoculated intravenously (IV), intraperiteoneally (IP), intramuscularly (IM), intranasally (IN), and subcutaneously (SubQ) with 5E6 Cn of a bioluminescent, luciferase-expressing strain of Cn (NE1269). At Days 0, 1, and 3, the brain, lungs, liver, spleen, kidneys, inguinal lymph nodes, and popliteal lymph nodes of each mouse 49Attorney Docket No.:049648-632156 UF Ref. T19361WO001 were excised and imaged via In Vivo Imaging System (IVIS) to track the bioluminescent Cn. The brain, lungs, liver, spleen, kidneys, inguinal lymph nodes, and popliteal lymph nodes were excised and imaged by bioluminescent imaging (results in Figure 7, Table 1). The total flux (photons / s) was used to display the relative bioluminescent signal for each organ. At Day 0, Cn was observed to accumulate within the lungs in both the IV and IN groups.24- and 72-hours post-inoculation, the bioluminescent signal for the IN group remained localized to the lungs while the IV group exhibited bioluminescent signals in the kidneys, spleen, lungs, and the brain with very minimal signal in the liver. These initial trends suggest the IV route is the most likely to result in Cn accumulation within the brain compared to the intranasal route. However, these trends, particularly the relative abundance of Cn in each organ, may differ in an ALS mouse with a dysregulated BBB which could facilitate more Cn accumulation in the brain.

[0228] The data demonstrate that Cn can disseminate to various organs throughout the body including the brain.

[0229] Protocol: Day 0: 1. Inoculation 2. Tissue Excision 3. Bioluminescent Imaging Day 1: 1. Tissue Excision 2. Bioluminescent Imaging Day 3: 1. Tissue Excision 2. Bioluminescent Imaging 50Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0230] Table 1: CN Accumulates in Specific Organs Based on Delivery Route Organ Intravenous Intraperitoneal Intranasal Intramuscular Delivery (Days Delivery (Days Delivery (Days Delivery (Daysadministration to healthy mice

[0232] Layer-By-Layer FDC Biodistribution Study

[0233] PLGA-NPs were linked by “click” chemistry to bioluminescent CN to produce bioluminescent FDCs (Figure 8) . 51Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0234] Quality Assurance of NP Conjugation to CN: Description: Before intravenous injection into healthy mice, FDCs were synthesized via the layer-by-layer formulation and characterized via flow cytometry for successful nanoparticle attachment.

[0235] The bioluminescent FDCs were delivered intravenously to healthy mice. Figure 8 is a schematic of layer-by-layer FDC biodistribution study. Brain, liver, spleen, and kidneys were excised on Days 0, 1, and 3, and analyzed by bioluminescent imaging. Figure 9 shows results as average radiance. Table 2 show results as proportional biodistribution.

[0236] Table 2. Layer-by-Layer FDCs Can Accumulate in the Brain Organ FDC Proportional FDC Proportional FDC Proportional Di ib i D 0 Di ib i D 1 Di ib i D 3

[0237] Example 15: Surface Engineering and Biodistribution of Fungal Drug Carriers

[0238] To generate the fungal carriers, the clinically-used material poly (lactic-co-glycolic) acid (PLGA) was used to form nanoparticles (Figures10A-10B) . The inventors assessed whether these nanoparticles could attach to the cryptococcal surface by pure adsorption through intermolecular forces or by forming a chemical bond with a functionalized version of PLGA (Figure 10A and Figure 18).

[0239] The PLGA particles were about 200-250nm in diameter and uniform in size according to their polydispersity indices (Figure 11A). Cryptococcal cells were roughly 5 micron in diameter (Figure 11B). The nanoparticles should be able to fit on the CN cell surface. 52Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0240] NPs and Cn have similar surface charges (Figure 12A). Similarly charged Cn and NP surfaces likely to result in repulsive Coulomb forces and reduced conjugation frequency (Figure 12B).

[0241] Figure 13 is a schematic depicting approaches for leveraging electrostatic interactions for NP attachment to Cn. (Top panel) Layer-by-Layer Approach; (Bottom panel) Single-layered Approach.

[0242] A Layer-By-Layer (LBL) Approach to FDC Synthesis is shown in Figure 14.

[0243] Figure 15 depicts LBL Approach Enables PLGA NP Adsorption to the Cn Surface

[0244] Description: Cn was pre-coated with PEI to enable electrostatic adsorption of negatively charged PLGA nanoparticles to the Cn surface. A color change was observed within the Cn pellet after incubating the PEI-coated Cn with the PLGA NPs, indicating successful attachment of the NPs. NP attachment was confirmed via flow cytometry.

[0245] Figure 16 depicts Quantifying NPs on Cn Surface

[0246] Conclusions: When aiming to attach 200 NPs to one Cn cell surface using the layer-by- layer method, ~22% of the particles successfully attach.

[0247] To assess stability, fungal carriers were incubated with serum proteins because when the fungal carriers are delivered to the body they will encounter a lot of different proteins. If the electrostatic interaction between the cell and the nanoparticle is stable, then the serum proteins should not be able to dislodge the nanoparticles from the surface. Figure 17 is a schematic of a serum test to evaluate coupling stability.

[0248] Figure 19 depicts results of studies showing that fungal carriers are viable and surface- bound particles are stably attached

[0249] Conclusions: After coating Cn in PEI, coupling PLGA NPs, and conjugating the NPs to the Cn surface, the resultant FDCs exhibited stable NP attachment and cell viability comparable to unmodified Cn (“Baseline”). Fungal carriers are viable and surface-bound NPs are stably attached. (A) Viability of CN unchanged as determined by PI staining. (B) summary of NP- 53Attorney Docket No.:049648-632156 UF Ref. T19361WO001 associated fluorescence of FDCs before and serum treatment. Serum is a fraction of the blood that contains a high concentration of proteins that are capable of interrupting interactions between NPs and Cn. Here, it is seen that in the presence of serum the Cn do not lose fluorescence from NPs previously attached.

[0250] Figure 20 results of studies showing FDCs can still execute vomocytosis.

[0251] Conclusions: After coating Cn in PEI, coupling PLGA NPs, and conjugating the NPs to the Cn surface, the resultant FDCs exhibited comparable rates of vomocytosis to unmodified Cn (“0:1”). Conjugation of NPs to the surface of Cn does not diminish the capacity of the Cn to induce vomocytosis from macrophages.

[0252] Figure 22 depicts results of experiments showing PEI Coating and Fungal Carrier Synthesis Result in Altered Surface Charges.

[0253] Conclusions: PLGA and PLGA-NHS NPs, and unmodified Cn exhibit negatively charged surfaces, which will likely cause repulsion and minimal coupling. When coated with the cationic polymer, PEI, Cn surfaces become slightly positive, and after attaching the negatively charged NPs to the PEI-coated Cn, the cell surface charge becomes negative once again. Zeta potential is a measure of the electrostatic potential at the slipping plane within the electrical double layer surrounding a dispersed particle in a colloidal system. Figure 22 shows that the surface charge is dramatically changed through the layering with PEI.

[0254] Figure 23 depicts results of experiments showing Surface Coating Cryptococcus neoformans with PEI Enables Electrostatic Adsorption of PLGA NPs.

[0255] Description: Cn was first coated with PEI, a cationic polymer, to tether negatively charged particles to negatively charged Cn. Using flow cytometry, PEI-coated Cn yields higher percentages of cells with at least one nanoparticle attached compared to unmodified Cn incubated with PLGA particles. A) Confocal fluorescent micrograph of an FDC. B) Flow cytometric analysis of FDC synthesis. Cells indicated as negative for nanoparticles did not have any surface-bound particles, whereas positive cells were observed to be decorated with nanoparticles. 54Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0256] Figure 24 depicts experiments showing PLGA Nanoparticles Remain Attached via Electrostatic Adsorption After Incubation with Serum Proteins.

[0257] Left-Hand Side Description: FDCs were incubated with serum proteins to determine the stability of the surface-bound particles for 1 hour. There were no significant changes (>20% reduction) in the percentage of number of Cn with at least one attached nanoparticle, nor were there significant changes (>50% reduction) in the overall number of particles on the Cn surface with or without a conjugation reaction.

[0258] Figure 25 depicts results of experiments showing Fungal drug carriers remain viable after synthesis.

[0259] Description: FDC viability was measured using a TexasRed-labeled propidium iodide viability dye. FDCs were compared to a heat-killed “Dead” control, and an unmodified control.

[0260] Figure 26 is a schematic showing Conjugation Schemes (EDC / NHS Reaction Strategy; Thiol-Maleimide Reaction Strategy; “Click” Chemistry”).

[0261] Figure 28 depicts results of experiments showing physicochemical properties of PLGA NPs.

[0262] Conclusions: Fabricated nanoparticles had an average hydrodynamic diameter (by number distribution) of ~250 nm and ~360 nm by volume distribution. NPs were negatively charged at their surface (~ - 23mV).

[0263] Figure 29 is a schematic showing a EDC / NHS Fungal Carrier Synthesis Scheme.

[0264] Figure 31 depicts results of experiments showing EDC / NHS Rxn: Particles Successfully Attached to CN.

[0265] Description: Acid-terminated PLGA nanoparticles were converted to NHS-terminated PLGA via EDC / NHS chemistry. The PLGA-NHS nanoparticles were then reacted with CFW- labeled Cn to conjugate the particles to the cell surface. 55Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0266] CFW-labeled Cn conjugated with PLGA-NHS nanoparticles were visualized by fluorescent microscopy. While ~48% of cells were positive for at least one particle, minimal surface decoration was observed on the cells such that most of the cell wall was visible. Less than 20% of CN had any NP associated fluorescence.

[0267] EDC / NHS Rxn: Particles Successfully Attached to CN, depicting micrographs. Conclusions: Acid-terminated PLGA particles functionalized via EDC / NHS reaction were able to attach to more Cn compared to ordinary non-functionalized PLGA particles, suggesting a conjugation-driven affinity towards the Cn surface.

[0268] Figure 32 is a schematic showing coating the NP with a Cationic Polymer.

[0269] Figure 34 depicts experiments showing PLL Increases Surface Charge of NPs.

[0270] Figures 35A-B depict results of experiments showing PLL Coating Increases NP Coupling to the CN Surface.

[0271] Cn were modified with PLGA nanoparticles using poly-l-lysine (PLL) and / or EDC / NHS chemistry. Cn were either modified with uncoated acid-terminated PLGA functionalized with NHS reactive groups, acid-terminated PLGA nanoparticles coated with PLL, or PLL-coated nanoparticles later functionalized with NHS reactive groups. Only PLL-coated, acid-terminated PLGA nanoparticles resulted in substantial attachment (>50% of Cn positive for at least one particle, >80% Cn had NP-associated fluorescence) to Cn via adsorption, suggesting that a positively charged polymer is key to facilitating surface modification. For PLL-coated nanoparticles that were later functionalized with NHS reactive groups via EDC / NHS chemistry, the NHS groups likely reacted with the free amines on PLL, thus reducing the surface charge of the particles. (Figure 35A) Flow cytometry data; (Figure 35B) Plot of NP+CN Coupling (Adsorbed vs. Conjugated) in the presence and absence of Poly-L-Lysine.

[0272] Figure 36 is a schematic showing potential conjugation of PLL to NPs may reduce the NP surface charge. (Left panel) PLL-coated PLGA-COOH NP; (Right panel) Expanded view showing 1. EDC-NHS-mediated ester functionalization of carboxyl group; 2. sNHS ester reaction with protonated PLL amines. 56Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0273] Figure 37 depicts results of experiments showing EDC / NHS reaction decreases PLL- coated NP surface charge.

[0274] PLL-Coated NPs Adsorb to CN Surface.

[0275] Images acquired during flow cytometry clearly show that nanoparticles coated with a cationic polymer (PLL or poly-L-lysine) result in surface-bound particles.

[0276] Figure 38 depicts results of experiments showing serum incubation results in significant loss of surface-bound NPs.

[0277] Figure 39 depicts results of experiments showing physicochemical Properties of PLGA- Conclusions: PLGA-MAL NPs are uniform in size as indicated by a PDI<0.2 and their surface charge can be modulated with PLL.

[0278] Figure 41 depicts experiments showing PLGA-MAL NPs Attach to CN, but Still Dislodge from CN Surface.

[0279] PLGA nanoparticles functionalized with maleimide (MAL) reactive groups, which may conjugate to the Cn surface via maleimide-thiol chemistry, were tested for conjugation to CN cells. At various ratios, NPs coated with poly-L-lysine adsorbed to the Cn surface. However, after incubating the modified Cn in serum, there was a considerable decrease in the percentage of cells positive for at least one particle, suggesting the PLGA-MAL particles do not successfully conjugate to most Cn surfaces.

[0280] Figure 42 depicts experiments showing MFI of CN+ for PLGA-MAL NPs Does not Shift Drastically.

[0281] Conclusions: When examining cells that are positive for at least one PLL-coated PLGA- MAL NP via flow cytometry, there is a lesser shift (>50%) in the number of particles attached to this positive population post-serum incubation. Stable conjugation of NPs to CN surface using maleimide-terminated PLGA-based NPs for varying ratios of NPs (mass) to CN number.

[0282] Figure 43 depicts results of experiments showing Fungal Carrier Viability is Ratio- Dependent for PLGA-MAL NP. 57Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0283] Conclusions: PLGA-MAL particles coated with PLL (~150kDa) exhibit toxic effects to Cn in a dose-dependent manner. CN viability is diminished as NP to CN ratio is increased.

[0284] Figure 44 is a schematic showing conjugation of PLGA-Maleimide NP to CN.

[0285] Figure 45 is a schematic showing conjugation of NPs and CN by “click” chemistry.

[0286] Example 16: Optimizing NP Loading on the Cn Surface

[0287] Synthesizing PLGA-PEI NPs. Figure 46A is a schematic showing how PLGA-PEI NPs were prepared and linked to CN using an adsorption approach.

[0288] Figure 46B is a schematic showing how PLGA-PEI-Azide NPs were prepared and linked to DBCO-functionalized CN using a conjugation approach.

[0289] Figure 46C is a schematic showing how PLGA-PEI-Azide NPs were synthesized.

[0290] Physicochemical Properties of PLGA-PEI NPs were characterized. Figure 47A shows plot of PLGA NP and PLGA-PEI-Azide NP dynamic light scattering. Figure 47B shows surface charge of fungal drug carrier components (CN, PLGA NPs, and PLGA-PEI-Azide NPs.

[0291] Observations in FDC Synthesis:

[0292] Description: Cn incubated with PLGA-PEI nanoparticles in low-sodium buffer tended to settle at the bottom of the incubation tube within 15 minutes.

[0293] CN were incubated with PLGA-PEI / Azide NPs. After centrifugation, the Cn cells appear to be decorated with the PLGA-PEI / Azide particles, indicated by the cell pellet’s color change.

[0294] Figure 49 shows results of experiments showing FDCs Efficiently and Stably Attach to the Cn Surface Without Compromising Intrinsic Functions

[0295] Fluorescent microscopy of FDCs synthesized at a 200:1, NP-to-Cn ratio showed that cationic PLGA nanoparticles overlay very well with the cell well, suggesting successful modification to the cell surface. 58Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0296] Description: PLGA-PEI particles functionalized with azide reactive groups remain attached to Cn functionalized with DBCO-reactive groups through “click” chemistry compared to non-functionalized Cn. There was a 3% reduction in FDCs formed through click chemistry, maintaining cryptococcal cell viability and comparable vomocytosis rates with unmodified cells. Figure 49A) Flow cytometry analysis of FDCs synthesized using Azide-DBCO “click” chemistry. Figure 49B) FDC viability assay. FDC viability was measured using a colony- forming unit (CFU) assay and was compared to heat-killed Cn. Figure 49C) FDC vomocytosis assay. FDC vomocytosis was measured using fluorescent, time-lapse microscopy.

[0297] Example 17: Characterizing Small Molecule Release from PLGA NPs at Varying pH

[0298] FDCs encounter dynamic environmental changes in vivo. Bloodstream pH is ~ 7.4, and phagosome pH at t=0.22 h is ~ 5.0 and phagosome pH at t=0.42 h is ~ 7.0

[0038] .

[0299] Characterizing the Effect of pH on PLGA NP Release Kinetics NP comprising Rhod-6G were prepared using PLGA of differing molecular weights and tested for release kinetics at pH 5.0 , pH, 7.0, and pH 7.4 5002A PLGA: 17kDa to prepare “LO” PLGA NP 5004A PLGA: 44kDa to prepare “MID” PLGA NP 5010 PLGA: 153kDa to prepare “HI” PLGA NP

[0300] Figure 50A-E show PLGA NP release profiles at varied pH. Figure 50A shows Rhod6g- loaded PLGA-PEI NP dynamic light scattering for NP prepared from 5002A, 5004A, and 5010 PLGA. Figure 50B shows Rhod-6g-loaded PLGA PEI-NP surface charge for NP prepared from 5002A, 5004A, and 5010 PLGA.

[0301] Figure 50C shows release of Rhod6G from NP prepared from 5002A PLGA, from 5004A PLGA, and from 5010 PLGA at pH 5 over time. Figure 50D shows release of Rhod6G from NP prepared from 5002A PLGA, from 5004A PLGA, and from 5010 PLGA at pH 7 over 59Attorney Docket No.:049648-632156 UF Ref. T19361WO001 time. Figure 50E shows release of Rhod6G from NP prepared from 5002A PLGA, from 5004A PLGA, and from 5010 PLGA at pH 7.4 over time.

[0302] Example 18: Evaluating the Effects of Varied UV Doses on Temporal CN Viability

[0303] Temporal CN Viability was measured in CN treated with varied UV doses.

[0304] Description of gating strategy: Cn viability data was characterized by first isolating single cells using a standard flow cytometry gating strategy.1) Using SSC-A vs FSC-A, all cells were selected.2) FSC-H vs FSC-A, single cells were selected.3) SSC-A vs CFW, cryptococcal cells positive for the cell wall dye were selected.4) Unviable cells were deselected for SSC-A vs. PI (TexasRed+) on the heat-killed control cells.

[0305] Description of “Untreated”: Cn that was not exposed to UV irradiation was used to establish the baseline cell viability.

[0306] Description: Cn was exposed to UV irradiation at various exposure times. Cn viability was measured over 6 days using TexasRed-labeled propidium iodide viability dye.

[0307] The method comprised: 1. Inoculate media with a colony of CN overnight (30°C, 150 rpm). 2. Dose CN with UV-C light (254 nm) for 2, 4, 6, and 8 days. 3. Incubate irradiated CN at 37 °C. 4. Measure CN viability via propidium iodide assay via flow cytometry at Day 0, Day 2, Day 4, Day 6, and Day 8.

[0308] Figure 51 shows UV-treated CN viability for untreated cells, Day 0, Day 1, Day 2, Day 4, and Day 6, for UV doses of 5 seconds, 10 seconds, 15 seconds, and 20 seconds. Conclusion: UV Irradiation Induces Time-dependent Reduction in Cn Viability.

[0309] Example 19: Intracellular Hydrogelation

[0310] To reduce in vivo replication of CN used in FDCs, CN is hydrogelated. To prepare hydrogelated CN, CN are combined with polyethylene glycol and a photoinitiator, for example 60Attorney Docket No.:049648-632156 UF Ref. T19361WO001 eosin-y, to first make CN infused with hydrogel monomers. The CN infused with hydrogel monomers are photoactivated by visible light exposure, to crosslink the intracellular hydrogel. Hydrogelated CN can be prepared for example as in Figure 52.

[0311] Example 20: Investigating Fungal Drug Carrier Efficacy in ALS

[0312] Figure 53 depicts schematic on Investigating Fungal Drug Carrier Efficacy in ALS. Riluzole and Edaravone loaded PLGA NPs are prepared and linked to tps1Δ CN to produce Riluzole+Edaravone-Loaded tps1Δ FDC. SOD1-G93A mouse model of ALS is treated with Riluzole+Edaravone-Loaded tps1Δ FDC. Survival, serum neurofilament levels, and persistence on a rotor rail are measured in SOD1-G93A mouse model of ALS treated with Riluzole+Edaravone-Loaded tps1Δ FDC. Endpoint characterization is performed via histology.

[0313] Example 21: PLGA NPs Adsorb Directly to the CN Polysaccharide Capsule

[0314] Figure 54 depicts scanning electron microscopy micrographs of an unmodified H99 cryptococcal cell and a H99 cryptococcal cell modified with PLGA nanoparticles on its surface. The PLGA nanoparticles appear to be directly and exclusively bound to the cell’s polysaccharide capsule with little to no adsorption to the cell wall.

[0315] Example 22: Systemic Immunosuppression and Varied Routes of Infection Influence Cryptococcus neoformans Trafficking Patterns and Organ Accumulation in Murine Models

[0316] Introduction

[0317] Cryptococcus neoformans (Cn) is an opportunistic fungal pathogen responsible for an estimated 220,000 infections and approximately 180,000 deaths, primarily due to cryptococcal meningitis. Cryptococcal meningitis occurs when Cn disseminates to the central nervous system, especially in individuals with compromised immune systems, such as those living with HIV. 61Attorney Docket No.:049648-632156 UF Ref. T19361WO001 Given its global burden and limited treatment options, the World Health Organization categorizes Cn as a “critical” threat to global health.

[0318] The treatment of cryptococcal infections faces many clinical and logistical hurdles. Amphotericin B, the gold standard of care, is associated with significant toxicity and remains largely inaccessible in regions where cryptococcal infection is most prevalent, such as Sub- Saharan Africa. Moreover, the antifungal pipeline is constrained by poor bioavailability, emerging resistance, and limited molecular targeting. Compounding these challenges is an incomplete understanding of how Cn disseminates from the initial site of infection to distant tissues, particularly across the blood-brain barrier (BBB), impeding the development of more targeted antifungal strategies.

[0319] Dissemination is a hallmark of Cn pathogenesis, and it has been categorized into three distinct processes thus far: receptor-mediated transcytosis, paracellular transport, and cell- mediated transport. The polysaccharide capsule of Cn, composed of glucuronoxylomannan and galactoxylomannan, is crucial for its virulence, facilitating interactions with host receptors, including CD14, CD44, RHAMM, and EphA2-tyrosine kinase receptors, on epithelial and endothelial cells. Disruption of capsule-receptor interactions significantly impairs Cn adhesion, uptake, and translocation, highlighting their importance in tissue invasion. Additionally, Cn can induce tissue damage through immune-mediated mechanisms. For instance, Cn stimulates IL-33 production in airway epithelial cells, downregulating expression of E-cadherin and ZO1 tight junction proteins, thus enabling paracellular migration.

[0320] Beyond extracellular trafficking, Cn can hijack phagocytes such as macrophages via a Trojan Horse-like mechanism to disseminate within the host. This intracellular mode of transport is hypothesized to facilitate Cn’s traversal across the BBB. Experimental models have demonstrated that Cn-loaded macrophages promote enhanced central nervous system colonization relative to free cryptococcal cells, and depletion of phagocytes reduces fungal burden in the brain and other organs. Although the Trojan Horse mechanism enhances Cn dissemination, numerous other mechanisms also critically contribute to its pathogenesis, particularly non-lytic exocytosis—termed "vomocytosis"—which enables Cn to escape from phagocytes. Vomocytosis has emerged as a potentially pivotal step in the onset of cryptococcal meningitis within the central nervous system. 62Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0321] Despite these insights, many questions remain about how host immunosuppression and the route of infection shape Cn dissemination. While HIV-associated immunosuppression is well established as a predisposing factor, emerging clinical evidence suggests that other forms of systemic immunosuppression, such as substance abuse or immunomodulatory therapies (e.g., JAK STAT inhibitors), may elevate risk. Furthermore, non-conventional transmission routes, including organ and tissue transplantation, have been implicated in clinical cases. These unconventional routes of infection, in conjunction with the recognized effects of immunosuppression, have not been systematically studied in the context of early Cn infection.

[0322] Previous research has examined the effects of immunosuppression and alternative routes of infection, although these have been analyzed separately and occasionally in conjunction with one another. However, these studies have primarily been constrained by models comparing healthy versus methamphetamine-treated mice and mice infected intranasally and intravenously. These approaches often assess a narrow set of organs and fail to reflect the complex interplay between exposure route, host status, and fungal tropism. A comprehensive, comparative analysis of how these factors independently and interactively influence dissemination dynamics is currently lacking.

[0323] Here, the inventors present a systematic study investigating the effects of infection route and systemic immunosuppression on the early dissemination of Cn in vivo. Using bioluminescent Cn and various inoculation routes—intranasal (IN), intraperitoneal (IP), intramuscular (IM), intravenous (IV), or subcutaneous (SubQ)— fungal dissemination was tracked over 72 hours in immunocompetent and tofacitinib (TFB)-treated mice, a clinically relevant model of JAK inhibitor-mediated immunosuppression. Dissemination was quantified across several organs, including the brain, lungs, heart, liver, spleen, pancreas, kidneys, and regional lymph nodes. Detailed histological and imaging analyses were performed on the brain, pancreas, spleen, and kidneys to characterize the dissemination patterns further.

[0324] Materials & Methods

[0325] Reagents YPD Agar (Sigma Aldrich, St. Louis, MO). YPD Broth (Sigma Aldrich, St. Louis, MO). Pierce™ D-Luciferin, Monopotassium Salt (ThermoFisher Scientific, Waltham, MA). 63Attorney Docket No.:049648-632156 UF Ref. T19361WO001 Tofacitinib Citrate (ThermoFisher Scientific, Waltham, MA).10% Formalin (Fisher Chemical, Pittsburgh, PA). Simvastatin

[0326] Animals Both male and female BALB / c mice aged 6 weeks were purchased from Jackson Laboratories. All animals were housed in an ABSL-2 facility at the University of Florida and used in accordance with the University of Florida Institutional Animal Care and Use Committee (Protocol No.0730).

[0327] C. neoformans Culture H99, luciferase-expressing, Cn was a generous gift from Greetje Vande Velde, Ph.D. Cn was grown on YPD agar plates for 72 hours at 30°C.24 hours before inoculation, a single colony of Cn was immersed in 7.5 mL of YPD broth supplemented with 2.5 mL of 40% glucose. The inoculated tube was placed in a floor incubator and left to shake at 150 RPM at 30°C overnight. Before inoculating mice, Cn were washed thrice via centrifugation at 3000 × g for 5 minutes in sterile 0.9% saline solution. The Cn were counted using a Cell Countess II FL (ThermoFisher Scientific, Waltham, MA) and diluted to the desired concentration in sterile 0.9% saline.

[0328] Tofacitinib Treatment for Induced Immunosuppression Some cohorts of mice were treated with 30 mg / kg of TFB each day for three days before inoculation. The mice received daily doses of TFB until they were sacrificed for tissue collection.

[0329] Simvastatin Treatment for Induced CD44 Depletion One cohort of mice were treated with 20 mg / kg of simvastatin each day for three days before inoculation. The mice received daily doses of simvastatin until they were sacrificed for tissue collection.

[0330] Bioluminescent Tracking of C. neoformans 5 × 106bioluminescent cryptococcal cells were administered to healthy and tofacitinib-treated cohorts of male and female BALB / c mice (6-9 weeks of age) via the following routes: IN, IV, IM, IP, or SubQ. IM and SubQ injections were administered to the right posterior biceps femoris and the lateral ventral region of the abdomen, respectively. Two hours, 24 hours, and 72 hours 64Attorney Docket No.:049648-632156 UF Ref. T19361WO001 post-inoculation, healthy and TFB-treated mice were injected IP with 150 mg / kg of Pierce™ D- Luciferin, Monopotassium Salt to generate a bioluminescent signal in vivo. Ten minutes post- luciferin injection, the mice were sacrificed via isoflurane overexposure, followed by cervical dislocation. The brain, lungs, heart, liver, spleen, pancreas, kidneys, and popliteal and inguinal lymph nodes (PLN and ILN) were excised and imaged using a Perkin Elmer IVIS CT In Vivo Imaging System (Waltham, MA). Respective tissues from saline control groups were included for background signal detection. Briefly, excised tissues were imaged using an exposure time of one minute, an F / stop of 1, and medium binning. The images were later analyzed using the Aura In Vivo Imaging Software (Spectral Instruments Imaging, Tucson, AZ) to measure the average radiance of each tissue (p / s / cm² / sr). The background signal measured from the corresponding saline tissues was subtracted to normalize the data. To minimize signal noise, the threshold to depict an arbitrarily colored bioluminescent signal was 1x104p / s / cm2 / sr.

[0331] Preparation of Histology Samples At Days 0, 1, and 3, mice from each cohort were sacrificed via CO2asphyxiation followed by cervical dislocation. Given the findings from the bioluminescence data, the brains, spleens, pancreases, and kidneys were harvested for further histological analysis. The excised tissues were incubated overnight in 10% Formalin at 4°C. The next day, the samples were washed twice with 1X Dulbecco’s Phosphate-Buffered Saline and stored in 70% ethanol. Samples were submitted to the University of Florida Molecular Pathology Core for processing, sectioning, and staining. Each tissue was sectioned at 50, 200, and 400µm and stained with Hematoxylin, Eosin (H&E), or Grocott’s methenamine silver (GMS).

[0332] Results

[0333] Routes of Infection Influence Cn Dissemination and Organ-Specific Accumulation in Healthy Mice

[0334] To investigate the effects of infection route on Cn trafficking patterns, five distinct infection routes of infection —IV, IN, IP, IM, and SubQ—were implemented and Cn dissemination was monitored across eight diverse tissues: brain, lungs, heart, liver, spleen, pancreas, kidneys, inguinal lymph nodes, and popliteal lymph nodes in murine models. A luciferase-expressing strain of Cn, developed by Vanherp et al.

[0035] was employed alongside 65Attorney Docket No.:049648-632156 UF Ref. T19361WO001 bioluminescent ex vivo imaging to elucidate Cn trafficking patterns; this technique provides superior sensitivity compared to conventional imaging modalities such as fluorescence.

[0335] The bioluminescent signals observed in various tissues were significantly influenced by the method of injection, highlighted by the average radiance measurements (photons / s / cm² / sr) for each tissue (Tables 4-15). Over a 72-hour observation period, the inventors identified substantial variations in bioluminescent signals across different tissues that correlated with the injection route (Tables 4-15). Each administration route yielded a distinct dissemination pattern of Cn within targeted tissues, suggesting potential organ-specific affinities (Tables 4-15). Mice undergoing intravenous (IV) injection displayed prominent bioluminescence in most excised tissues, including the brain, lungs, heart, liver, spleen, pancreas, and kidneys. On Day 0, 87.48% of the total bioluminescence was concentrated in the lungs, which subsequently declined to 15.49% and 10.93% by Days 1 and 3, respectively (Figure 56B, Tables 3A-C). This pattern likely reflects the first-pass effect in the lungs following tail-vein injection. Furthermore, IV- delivered Cn showed a pronounced tendency to accumulate in the brain compared to other routes of infection, underscoring the critical role of fungemia in the pathogenesis of cryptococcal meningitis (Figure 56A, Tables 3A-C).

[0336] In contrast, IP injection led to significant Cn accumulation within the pancreas, which accounted for 53.6% of the total bioluminescence, outpacing all other injection routes (Figure 56F, Table 1, Table 4). When Cn was administered intranasally in healthy mice, the cryptococcal cells largely remained contained within the lungs, likely due to a robust pulmonary immune response characterized by mechanisms such as phagocytosis and granuloma formation. Similarly, SubQ injections in healthy mice primarily displayed bioluminescent signals localized to the injection site. For example, an IVIS bioluminescence image of a mouse infected subcutaneously with Cn at Day 3, showed that bioluminescent signal was prominent at the injection site, suggesting the cryptococcal cells remain localized and do not tend to distribute to other tissues. While the normalized average radiance values for the tissues excised from SubQ-infected mice are non-zero and exhibit trends, they remain below the color threshold set to minimize noise. Cryptococcal cells delivered IM migrated to the draining lymph nodes, particularly the right PLN, and to the contralateral ILN (Tables 4-15). Disseminated cryptococcal cells showed a marked preference for accumulation in the right PLN, with 66.86% of the bioluminescent signal originating from this site 66Attorney Docket No.:049648-632156 UF Ref. T19361WO001 compared to 19.89% from the left ILN (Table 1). Furthermore, a significant decrease in bioluminescence in the right PLN over time was observed, possibly indicating a resolution mediated by lymph node-resident antigen-presenting cells (Figure 56I, Table 3A-C).

[0337] Tofacitinib-Mediated Systemic Immunosuppression Alters Cn Dissemination Patterns Observed in Healthy Mice

[0338] A comprehensive investigation was conducted into the effects of systemic immunosuppression on Cn dissemination, utilizing TFB—a JAK-STAT inhibitor designed to attenuate immune responses in autoimmune disorders—in a cohort of mice subjected to IV, IN, IP, IM, or SubQ-mediated infections (Figure 55). Cn dissemination exhibited significant variations contingent upon the injection route (Tables 16-32). The dissemination patterns were also heavily influenced by the status of the immune system (Figure 56A-K , Table 3A-C, Tables 28-32). TFB-treated mice undergoing IN or IM infection exhibited the most pronounced dynamics in Cn dissemination compared to their healthy counterparts, which were observable as early as Day 0, only hours post-infection. A substantial portion of the bioluminescent signal in TFB-treated IN and IM-infected mice was traced back to the pancreas, accounting for 44% and 60.72%, respectively. Furthermore, log2-fold changes (Log2FC) greater than 1 were observed across the right ILN, left PLN, lungs, heart, liver, spleen, pancreas, kidneys, and brain of TFB- treated, IM-infected mice, indicative of a marked shift in Cn trafficking compared to the healthy cohort (Tables 33-35). TFB-treated, IN-infected mice mirrored these trends, with Log2FC>1 observed in the heart, liver, spleen, pancreas, kidneys, right ILN, and left PLN. Collectively, these alterations in Cn trafficking suggest that the suppressed local immune environments are unable to curb the infection, in stark contrast to observations in healthy mice.

[0339] Systemic administration of Cn via IV or IP injection routes resulted in distinct alterations in tissue trafficking patterns following treatment with TFB. Notably, analysis of livers, spleens, pancreases, and kidneys from IV-infected, TFB-treated mice revealed significant reductions in Cn trafficking, characterized by Log2FC values of less than -1 (Tables 33-35). Conversely, tissues, including hearts, lungs, and PLNs, from IP-infected, TFB-treated mice exhibited enhanced Cn trafficking, as indicated by Log2FC values greater than 1.

[0340] Table 3. Proportional Biodistribution Based on Bioluminescence Signal Acquired via IVIS, Day 0, Day 1, Day 3 67

[0341] Table 3A: Proportional Biodistribution Based on Bioluminescence Signal Acquired via IVIS (Day 0)Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0342] Table 3B Proportional Biodistribution Based on Bioluminescence Signal Acquired via IVIS (Day 1) IV IP IN IM SubQ* Tissue Healthy TFB Healthy TFB Healthy TFB Healthy TFB Healthy TFB i % % % % % % % % % % %Acquired via IVIS (Day 3) IV IP IN IM SubQ* Tissue Healthy TFB Healthy TFB Healthy TFB Healthy TFB Healthy TFB % % % % % % % % % % %69Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0344] Table 4. Effect of Injection Route on Cn Trafficking in Healthy Mice. Statistical Test: Ordinary Two-Way ANOVA, Significance: p≤0.05 (bolded). Tissue P Value P Value P Value (Injection Route) (Time) (Injection*Time

[0345] Table 5. Effect of Injection Route on Cn Trafficking to the Brain in Healthy Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Brain Comparison Adjusted P Value IV vs IP 0799970Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0346] Table 6. Effect of Injection Route on Cn Trafficking to the Lungs in Healthy Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Lungs Comparison Adjusted P Value IV vs. IP 0.0213

[0347] Table 7. Effect of Injection Route on Cn Trafficking to the Heart in Healthy Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Heart Comparison Adjusted P Value IV vs. IP 0.000371Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0348] Table 8. Effect of Injection Route on Cn Trafficking to the Liver in Healthy Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Liver Comparison Adjusted P Value IV vs. IP 0.9993

[0349] Table 9. Effect of Injection Route on Cn Trafficking to the Spleen in Healthy Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Spleen Comparison Adjusted P Value IV vs. IP <0.000172Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0350] Table 10. Effect of Injection Route on Cn Trafficking to the Pancreas in Healthy Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Pancreas Comparison Adjusted P Value IV vs. IP <0.0001

[0351] Table 11. Effect of Injection Route on Cn Trafficking to the Kidneys in Healthy Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Kidneys Comparison Adjusted P Value IV vs. IP <0.000173Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0352] Table 12. Effect of Injection Route on Cn Trafficking to the Right ILN in Healthy Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Right ILN Comparison Adjusted P Value IV vs. IP 0.9999

[0353] Table 13. Effect of Injection Route on Cn Trafficking to the Right PLN in Healthy Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Right PLN Comparison Adjusted P Value IV vs. IP >0.999974Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0354] Table 14. Effect of Injection Route on Cn Trafficking to the Left ILN in Healthy Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Left ILN Comparison Adjusted P Value

[0355] Table 15. Effect of Injection Route on Cn Trafficking to the Left PLN in Healthy Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Left PLN Comparison Adjusted P Value IV vs. IP 0.193375Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0356] Table 16. Effect of Injection Route on Cn Trafficking in TFB-Treated Mice. Statistical Test: Ordinary Two-Way ANOVA, Significance: p≤0.05 (bolded). Tissue P Value P Value P Value (Injection Route) (Time) (Injection*Time

[0357] Table 17. Effect of Injection Route on Cn Trafficking to the Brain in TFB-Treated Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Brain Comparison Adjusted P Value IV vs. IP <0.000176Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0358] Table 18. Effect of Injection Route on Cn Trafficking to the Lungs in TFB-Treated Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Lungs Comparison Adjusted P Value IV vs. IP <0.0001

[0359] Table 19. Effect of Injection Route on Cn Trafficking to the Heart in TFB-Treated Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Heart Comparison Adjusted P Value IV vs. IP 0.994477Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0360] Table 20. Effect of Injection Route on Cn Trafficking to the Liver in TFB-Treated Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Liver Comparison Adjusted P Value IV vs. IP 0.0003

[0361] Table 21. Effect of Injection Route on Cn Trafficking to the Spleen in TFB-Treated Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Spleen Comparison Adjusted P Value IV vs. IP <0.000178Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0362] Table 22. Effect of Injection Route on Cn Trafficking to the Pancreas in TFB-Treated Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Pancreas Comparison Adjusted P Value IV vs. IP <0.0001

[0363] Table 23. Effect of Injection Route on Cn Trafficking to the Kidneys in TFB-Treated Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Kidneys Comparison Adjusted P Value IV vs. IP 0.000179Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0364] Table 24. Effect of Injection Route on Cn Trafficking to the Right ILN in TFB-Treated Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Right ILN Comparison Adjusted P Value IV vs. IP 0.5720

[0365] Table 25. Effect of Injection Route on Cn Trafficking to the Right PLN in TFB-Treated Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Right PLN Comparison Adjusted P Value IV vs. IP 0.998480Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0366] Table 26. Effect of Injection Route on Cn Trafficking to the Left ILN in TFB-Treated Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Left ILN Comparison Adjusted P Value

[0367] Table 27. Effect of Injection Route on Cn Trafficking to the Left PLN in TFB-Treated Mice (Multiple Comparisons) Post-Hoc Test: Tukey’s, Significance: p≤0.05 (bolded). Left PLN Comparison Adjusted P Value IV vs. IP 0.234281Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0368] Table 28. Effect of TFB Treatment on Cn Trafficking During IV Infection (Healthy vs TFB). Statistical Test: Ordinary Two-Way ANOVA, Significance: p≤0.05 (bolded). IV Infection Tissue P Value P Value P Value (Condition) (Time) (Condition*Time

[0369] Table 29. Effect of TFB Treatment on Cn Trafficking During IP Infection (Healthy vs TFB). Statistical Test: Ordinary Two-Way ANOVA, Significance: p≤0.05 (bolded). IP Infection Tissue P Value P Value P Value (Condition) (Time) (Condition*Time82Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0370] Table 30. Effect of TFB Treatment on Cn Trafficking During IN Infection (Healthy vs TFB). Statistical Test: Ordinary Two-Way ANOVA, Significance: p≤0.05 (bolded). IN Infection Tissue P Value P Value P Value (Condition) (Time) (Condition*Time

[0371] Table 31. Effect of TFB Treatment on Cn Trafficking During IM Infection (Healthy vs TFB). Statistical Test: Ordinary Two-Way ANOVA, Significance: p≤0.05 (bolded). IM Infection Tissue P Value P Value P Value (Condition) (Time) (Condition*Time83Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0372] Table 32. Effect of TFB Treatment on Cn Trafficking During SubQ Infection (Healthy vs TFB). Statistical Test: Ordinary Two-Way ANOVA, Significance: p≤0.05 (bolded). SubQ Infection Tissue P Value P Value P Value (Condition) (Time) (Condition*Time84

[0373] Table 33 Log Two-Fold Changes in Cn Trafficking After TFB Treatment at Day 0.Formula:Day 0Attorney Docket No.:049648-632156 UF Ref. T19361WO001 Tissue Average Radiance Average Radiance LOG2FC (Healthy) (TFB)86

[0374] Table 34. Log Two-Fold Changes in Cn Trafficking After TFB Treatment at Day 1.Formula:Day 1Attorney Docket No.:049648-632156 UF Ref. T19361WO001 Tissue Average Radiance Average Radiance LOG2FC (Healthy) (TFB)88

[0375] Table 35 Log Two-Fold Changes in Cn Trafficking After TFB Treatment at DayFormula:Day 3Attorney Docket No.:049648-632156 UF Ref. T19361WO001 Tissue Average Radiance Average Radiance LOG2FC (Healthy) (TFB)Simvastatin-Mediated CD44 Depletion Reduces Cn Accumulation within Tissues CD44 is a ubiquitously expressed receptor found in every tissue and has been implicated in facilitating Cn dissemination to the brain. CD44 depletion significantly reduces Cn dissemination to the brain

[0036] . Based on the current understanding of CD44-Cn interactions coupled with our bioluminescence data, the inventors tested whether CD44 depletion affects Cn dissemination to other tissues. Mice were pre-treated with simvastatin, a cholesterol-modulating medication shown to disrupt lipid rafts in which CD44 resides in cell membranes, and subsequently infected intravenously with bioluminescent Cn. The same organs of interest were excised and imaged via IVIS. At as early as Day 0, there were significant decreases in bioluminescent signal in infected mice treated with simvastatin compared to untreated, infected mice (Figure 57A-K). This trend was observed across all tissues, suggesting CD44 may be critical in Cn dissemination into all tissues, and not exclusively the brain. 90Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0376] Example 23: MZ-101 Loaded FDCs

[0377] MZ-101-loaded FDCs were prepared. Figure 58 is a schematic of MZ101-Loaded PLGA Nanoparticle Synthesis

[0378] The method comprised: 5. Oil-in-water emulsification of organic phase (dichloromethane as solvent, PLGA (100mg) +3 mg PEI + MZ101 (0.5 mg) and aqueous phase (10% poly-vinyl alcohol) 6. Nanoparticles formed using homogenization (25k RPM for 3 minutes). 7. DCM solvent evaporation overnight in 2.5% PVA solution. 8. Particles washed via centrifugation in DI H2O and lyophilized.

[0379] Figure 59 depicts MZ101 NP Physicochemical Properties. (left panel) MZ101 NPs Dynamic Light Scattering; (right panel) Surface Charge of FDC Components. Figure 60 depicts Calculating MZ101 Loading into Nanoparticles. (left panel): Fluorescent MZ101 Standard Curve; (right panel) Tabulated data

[0380] Example 24: Gemcitabine-Loaded FDCs

[0381] Gemcitabine-Loaded FDCs were prepared. Figure 61 is a schematic of Gemcitabine- Loaded PLGA Nanoparticle (GNP) Synthesis.

[0382] The method comprised: 1. 4 mg of Gemcitabine hydrochloride (0.2 mL) added dropwise to 100 mg / mL PLGA + 3 mg / mL PEI + dichloromethane solution (4mL) 2. Homogenized the first emulsion at 25kRPM for 1 minute. 3. First emulsion added dropwise to 10%V PVA solution. 4. Homogenized the second emulsion at 25kRPM for 3 minutes. 91Attorney Docket No.:049648-632156 UF Ref. T19361WO001 5. Stirred the particles in 2.5% PVA overnight to evaporate the DCM solvent.

[0383] Figure 62 depicts GNP Physicochemical Properties. (left panel) Gemcitabine-Loaded PLGA NPs Dynamic Light Scattering; (right panel) Surface Charge of Gemcitabine-Loaded NPs. Figure 63 depicts Calculating Gemcitabine Loading into Nanoparticles. (left panel) Gemcitabine Standard Curve; (right panel) Tabulated data. 92Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0384] References [1] J. Van Schependom and M. D’haeseleer, “Advances in Neurodegenerative Diseases,” JCM, vol.12, no.5, p.1709, Feb.2023, doi: 10.3390 / jcm12051709. [2] D. Wu, Q. Chen, X. Chen, F. Han, Z. Chen, and Y. Wang, “The blood–brain barrier: structure, regulation, and drug delivery,” Sig Transduct Target Ther, vol.8, no.1, p.217, May 2023, doi: 10.1038 / s41392-023-01481-w. [3] L. Tzeplaeff, S. Wilfling, M. V. Requardt, and M. Herdick, “Current State and Future Directions in the Therapy of ALS,” Cells, vol.12, no.11, p.1523, May 2023, doi: 10.3390 / cells12111523. [4] “Riluzole | C8H5F3N2OS | CID 5070 - PubChem”. [5] “Edaravone | C10H10N2O | CID 4021 - PubChem”. [6] R. S. Kadam, D. W. A. Bourne, and U. B. Kompella, “Nano-Advantage in Enhanced Drug Delivery with Biodegradable Nanoparticles: Contribution of Reduced Clearance,” Drug Metab Dispos, vol.40, no.7, pp.1380–1388, Jul.2012, doi: 10.1124 / dmd.112.044925. [7] G. Y. Wang, S. L. Rayner, R. Chung, B. Y. Shi, and X. J. Liang, “Advances in nanotechnology-based strategies for the treatments of amyotrophic lateral sclerosis,” Materials Today Bio, vol.6, p.100055, Mar.2020, doi: 10.1016 / j.mtbio.2020.100055. [8] B. Sethi, V. Kumar, K. Mahato, D. W. Coulter, and R. I. Mahato, “Recent advances in drug delivery and targeting to the brain,” Journal of Controlled Release, vol.350, pp.668–687, Oct.2022, doi: 10.1016 / j.jconrel.2022.08.051. [9] R. Rampado, S. Crotti, P. Caliceti, S. Pucciarelli, and M. Agostini, “Recent Advances in Understanding the Protein Corona of Nanoparticles and in the Formulation of ‘Stealthy’ Nanomaterials,” Front. Bioeng. Biotechnol., vol.8, p.166, Apr.2020, doi: 10.3389 / fbioe.2020.00166.

[0010] S. Denham and J. Brown, “Mechanisms of Pulmonary Escape and Dissemination by Cryptococcus neoformans,” JoF, vol.4, no.1, p.25, Feb.2018, doi: 10.3390 / jof4010025.

[0011] Y. C. Chang et al., “Cryptococcal Yeast Cells Invade the Central Nervous System via Transcellular Penetration of the Blood-Brain Barrier,” INFECT. IMMUN., vol.72, p.11, 2004.

[0012] C. Charlier, K. Nielsen, S. Daou, M. Brigitte, F. Chretien, and F. Dromer, “Evidence of a Role for Monocytes in Dissemination and Brain Invasion by Cryptococcus neoformans,” Infect Immun, vol.77, no.1, pp.120–127, Jan.2009, doi: 10.1128 / IAI.01065-08.

[0013] Y. Zhou et al., “The pathways and the mechanisms by which Cryptococcus enters the brain,” Mycology, pp.1–15, Feb.2024, doi: 10.1080 / 21501203.2023.2295409.

[0014] X. Yang, H. Wang, F. Hu, X. Chen, and M. Zhang, “Nonlytic exocytosis of Cryptococcus neoformans from neutrophils in the brain vasculature,” Cell Commun Signal, vol.17, no.1, p.117, Dec.2019, doi: 10.1186 / s12964-019-0429-0.

[0015] T. B. Kechichian, J. Shea, and M. Del Poeta, “Depletion of Alveolar Macrophages Decreases the Dissemination of a Glucosylceramide-Deficient Mutant of Cryptococcus neoformans in Immunodeficient Mice,” Infect Immun, vol.75, no.10, pp.4792–4798, Oct. 2007, doi: 10.1128 / IAI.00587-07.

[0016] K. Kaufman-Francis et al., “The Early Innate Immune Response to, and Phagocyte- Dependent Entry of, Cryptococcus neoformans Map to the Perivascular Space of Cortical Post-Capillary Venules in Neurocryptococcosis,” The American Journal of Pathology, vol. 188, no.7, pp.1653–1665, Jul.2018, doi: 10.1016 / j.ajpath.2018.03.015. 93Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0017] D. Patel et al., “Methamphetamine Enhances Cryptococcus neoformans Pulmonary Infection and Dissemination to the Brain,” mBio, vol.4, no.4, pp. e00400-13, Aug.2013, doi: 10.1128 / mBio.00400-13.

[0018] E. A. Eugenin, J. M. Greco, S. Frases, J. D. Nosanchuk, and L. R. Martinez, “Methamphetamine Alters Blood Brain Barrier Protein Expression in Mice, Facilitating Central Nervous System Infection by Neurotropic Cryptococcus neoformans,” The Journal of Infectious Diseases, vol.208, no.4, pp.699–704, Aug.2013, doi: 10.1093 / infdis / jit117.

[0019] K. Goughenour et al., “Cryptococcus neoformans trehalose-6-phosphate synthase (tps1) promotes organ-specific virulence and fungal protection against multiple lines of host defenses,” Front. Cell. Infect. Microbiol., vol.14, p.1392015, May 2024, doi: 10.3389 / fcimb.2024.1392015.

[0020] R. Cheng and S. Wang, “Cell-mediated nanoparticle delivery systems: towards precision nanomedicine,” Drug Deliv. and Transl. Res., Apr.2024, doi: 10.1007 / s13346-024-01591- 0.

[0021] N. L. Klyachko et al., “Macrophages with cellular backpacks for targeted drug delivery to the brain,” Biomaterials, vol.140, pp.79–87, Sep.2017, doi: 10.1016 / j.biomaterials.2017.06.017.

[0022] N. Kapate et al., “A backpack-based myeloid cell therapy for multiple sclerosis,” Proc. Natl. Acad. Sci. U.S.A., vol.120, no.17, p. e2221535120, Apr.2023, doi: 10.1073 / pnas.2221535120.

[0023] N. Kapate et al., “Backpack-mediated anti-inflammatory macrophage cell therapy for the treatment of traumatic brain injury,” PNAS Nexus, p. pgad434, Dec.2023, doi: 10.1093 / pnasnexus / pgad434.

[0024] C. J. Bashor, I. B. Hilton, H. Bandukwala, D. M. Smith, and O. Veiseh, “Engineering the next generation of cell-based therapeutics,” Nat Rev Drug Discov, vol.21, no.9, pp.655– 675, Sep.2022, doi: 10.1038 / s41573-022-00476-6.

[0025] A. M. Vargason and A. C. Anselmo, “Clinical translation of microbe‐based therapies: Current clinical landscape and preclinical outlook,” Bioengineering & Transla Med, vol.3, no.2, pp.124–137, May 2018, doi: 10.1002 / btm2.10093.

[0026] T. C. Larson, W. Kaye, P. Mehta, and D. K. Horton, “Amyotrophic Lateral Sclerosis Mortality in the United States, 2011–2014,” Neuroepidemiology, vol.51, no.1–2, pp.96– 103, 2018, doi: 10.1159 / 000488891.

[0027] D. Petrov, C. Mansfield, A. Moussy, and O. Hermine, “ALS Clinical Trials Review: 20 Years of Failure. Are We Any Closer to Registering a New Treatment?,” Front. Aging Neurosci., vol.9, Mar.2017, doi: 10.3389 / fnagi.2017.00068.

[0028] H. Lu, W. Dong Le, Y.-Y. Xie, and X.-P. Wang, “Current Therapy of Drugs in Amyotrophic Lateral Sclerosis,” CN, vol.14, no.4, pp.314–321, Apr.2016, doi: 10.2174 / 1570159X14666160120152423.

[0029] B. Xiong et al., “Strategies for Structural Modification of Small Molecules to Improve Blood–Brain Barrier Penetration: A Recent Perspective,” J. Med. Chem., vol.64, no.18, pp.13152–13173, Sep.2021, doi: 10.1021 / acs.jmedchem.1c00910.

[0030] S. Garbuzova-Davis et al., “Impaired blood–brain / spinal cord barrier in ALS patients,” Brain Research, vol.1469, pp.114–128, Aug.2012, doi: 10.1016 / j.brainres.2012.05.056.

[0031] R. Mészáros, I. Varga, and T. Gilányi, “Adsorption of Poly(ethyleneimine) on Silica Surfaces: Effect of pH on the Reversibility of Adsorption,” Langmuir, vol.20, no.12, pp. 5026–5029, Jun.2004, doi: 10.1021 / la049611l. 94Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0032] E. J. Santiago-Burgos, P. V. Stuckey, and F. H. Santiago-Tirado, “Real-time visualization of phagosomal pH manipulation by Cryptococcus neoformans in an immune signal- dependent way,” Front. Cell. Infect. Microbiol., vol.12, p.967486, Sep.2022, doi: 10.3389 / fcimb.2022.967486.

[0033] V. Kozlovskaya et al., “Hydrogen-bonded LbL shells for living cell surface engineering,” Soft Matter, vol.7, no.6, pp.2364–2372, 2011, doi: 10.1039 / C0SM01070G.

[0034] G. Wang et al., “Decorating an individual living cell with a shell of controllable thickness by cytocompatible surface initiated graft polymerization,” Chem. Commun., vol.54, no.37, pp.4677–4680, 2018, doi: 10.1039 / C8CC01311J.

[0035] L. Vanherp et al., “Sensitive bioluminescence imaging of fungal dissemination to the brain in mouse models of cryptococcosis,” Disease Models & Mechanisms, p. dmm.039123, Jan. 2019, doi: 10.1242 / dmm.039123.

[0036] A. M. Al-Huthaifi, B. A. Radman, A. A. Al-Alawi, F. Mahmood, and T.-B. Liu, “Mechanisms and Virulence Factors of Cryptococcus neoformans Dissemination to the Central Nervous System,” JoF, vol.10, no.8, p.586, Aug.2024, doi: 10.3390 / jof10080586.

[0044] Sun D, et al., Why 90% of clinical drug development fails and how to improve it? Acta Pharm Sin B.2022 Jul;12(7):3049-3062

[0045] Rosenblum D, et al., Progress and challenges towards targeted delivery of cancer therapeutics. Nat Commun.2018 Apr 12;9(1):1410.

[0046] Kress H, et al., Cell stimulation with optically manipulated microsources. Nat Methods. 2009 Dec;6(12):905-9.

[0047] Gong C, et al., Harnessing the heterogeneity of T cell differentiation fate to fine-tune generation of effector and memory T cells. Front Immunol.2014 Feb 19;5:57.

[0048] Nicola AM, et al., 2011. Nonlytic Exocytosis of Cryptococcus neoformans from Macrophages Occurs In Vivo and Is Influenced by Phagosomal pH. Bio 2:10.1128 / mbio.00167- 11.

[0049] Rajasingham R, et al., The global burden of HIV-associated cryptococcal infection in adults in 2020: a modelling analysis. Lancet Infect Dis.2022 Dec;22(12):1748-1755.

[0050] Sabiti.W. and May R.C., Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans Future Microbiol., 2012, 7(11):1-17

[0051] Stephan MT, Moon JJ, Um SH, Bershteyn A, Irvine DJ. Therapeutic cell engineering with surface-conjugated synthetic nanoparticles. Nat Med.2010 Sep;16(9):1035-41.

[0052] Marshall GP, Cserny J, Wang CW, Looney B, Posgai AL, Bacher R, Keselowsky B, Brusko TM. Biomaterials-based nanoparticles conjugated to regulatory T cells provide a modular system for localized delivery of pharmacotherapeutic agents. J Biomed Mater Res A. 2023 Feb;111(2):185-197

[0053] rakash S, Kumbhojkar N, Lu A, Kapate N, Suja VC, Park KS, Wang LL, Mitragotri S. Polymer Micropatches as Natural Killer Cell Engagers for Tumor Therapy. ACS Nano.2023 Aug 22;17(16):15918-15930.

[0054] Brenner, J.S., Pan, D.C., Myerson, J.W. et al. Red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magnitude. Nat Commun 9, 2684 (2018).

[0055] N. Kapate, et al., A backpack-based myeloid cell therapy for multiple sclerosis, Proc. Natl. Acad. Sci. U.S.A.120 (17) e2221535120, 95Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0056] Nosanchuk JD, and Casadevall A. Cellular charge of Cryptococcus neoformans: contributions from the capsular polysaccharide, melanin, and monoclonal antibody binding. Infect Immun.1997 May;65(5):1836-41.

[0057] Gonçalves DS, et al., Histoplasma capsulatum Glycans From Distinct Genotypes Share Structural and Serological Similarities to Cryptococcus neoformans Glucuronoxylomannan. Front Cell Infect Microbiol.2021 Jan 8;10:565571.

[0058] Wang, Li, and Doering, Glycobiology.2018 Oct; 28(10): 719–730

[0059] Marshall GP, et al., Biomaterials-based nanoparticles conjugated to regulatory T cells provide a modular system for localized delivery of pharmacotherapeutic agents. J Biomed Mater Res A.2023 Feb;111(2):185-197.

[0060] Tang L, et al., Enhancing T cell therapy through TCR-signaling-responsive nanoparticle drug delivery. Nat Biotechnol.2018 Sep;36(8):707-716.

[0061] Wikipedia “Cryptococcus neoformans” World Wide Web at en.wikipedia.org / w / index.php?title=Cryptococcus_neoformans&oldid=1223677857

[0062] Hamed, M. F. et al., mBio.2023 Apr 25;14(2):e0264022

[0063] Allen et al., ACS Appl. Bio Mater.2019, 2, 2388−2404

[0064] Devaraj & Finn Chem. Rev.2021, 121, 12, 6697–6698

[0065] Lin, JC., et al., Intracellular hydrogelation preserves fluid and functional cell membrane interfaces for biological interactions. Nat Commun 10, 1057 (2019).

[0066] Wang, J. et al., Intracellular hydrogelation of macrophage conjugated probiotics for hitchhiking delivery and combined treatment of colitis, Materials Today Bio, 20, 100679 (2023),

[0067] US20250034519A1 HYDROGELATED CELLS, January 30, 2025.

[0068] Baghdasaryan, O., Contreras-Llano, L.E., Khan, S. et al. Fabrication of cyborg bacterial cells as living cell–material hybrids using intracellular hydrogelation. Nat Protoc 19, 3613–3639 (2024). https: / / doi.org / 10.1038 / s41596-024-01035-6

[0069] Wang Y, Casadevall A. Decreased susceptibility of melanized Cryptococcus neoformans to UV light. Appl Environ Microbiol.1994 Oct;60(10):3864-6;

[0070] Bryan R, Jiang Z, Friedman M, Dadachova E. The effects of gamma radiation, UV and visible light on ATP levels in yeast cells depend on cellular melanization. Fungal Biol.2011 Oct;115(10):945-9;

[0071] Schiave LA, Pedroso RS, Candido RC, Roberts DW, Braga GU. Variability in UVB tolerances of melanized and nonmelanized cells of Cryptococcus neoformans and C. laurentii. Photochem Photobiol.2009 Jan-Feb;85(1):205-13.

[0072] Martinez LR, Casadevall A. Cryptococcus neoformans biofilm formation depends on surface support and carbon source and reduces fungal cell susceptibility to heat, cold, and UV light. Appl Environ Microbiol.2007 Jul;73(14):4592-601;

[0073] Jacobson ES, Ayers DJ, Harrell AC, Nicholas CC. Genetic and phenotypic characterization of capsule mutants of Cryptococcus neoformans. J Bacteriol.1982 Jun;150(3):1292-6

[0074] Xu Y, et al., CD44 and its implication in neoplastic diseases. MedComm (2020).2024 May 23;5(6):e554..

[0075] Krolikoski, M. et al., The CD44-HA axis and inflammation in atherosclerosis: A temporal perspective, Matrix Biology, Volumes 78–79, 2019, Pages 201-218

[0076] Rousseau, D. et al., CD44 in myeloid cells is a major driver of liver inflammation and injury in alcohol-associated liver disease. Hepatology, January 14, 2025. |

[0077] Chen, L. et al., The role of CD44 in pathological angiogenesis The FASEB Journal 34(10) 13125-13139. 96Attorney Docket No.:049648-632156 UF Ref. T19361WO001

[0385] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0386] All publications, databases, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference. 97

Claims

Attorney Docket No.:049648-632156 UF Ref. T19361WO001 WHAT IS CLAIMED IS:

1. A fungal drug carrier composition comprising an avirulent fungal pathogen cell and at least one drug-loaded nanoparticle (NP) linked to the surface of the avirulent fungal pathogen cell, wherein the avirulent fungal pathogen cell is coated with a first cationic polymer.

2. A fungal drug carrier composition comprising an avirulent fungal pathogen cell and at least one drug-loaded nanoparticle (NP) linked to the surface of the avirulent fungal pathogen cell, wherein the at least one drug-loaded nanoparticle (NP) comprises a first cationic polymer.

3. The fungal drug carrier of claim 1 or claim 2, wherein the avirulent fungal pathogen cell is Cryptococcus or Candida albicans..

4. The fungal drug carrier of claim 3, wherein the avirulent fungal pathogen cell is Cryptococcus neoformans , Cryptococcus gatti, or Cryptococcus grubii.

5. The fungal drug carrier of claim 4, wherein the avirulent fungal pathogen cell is Cryptococcus neoformans.

6. The fungal drug carrier of claim 5, wherein the avirulent fungal pathogen cell is Cryptococcus neoformans H99, Cryptococcus neoformans tps1Δ, or Cryptococcus neoformans plb1.

7. The fungal drug carrier of any previous claim, wherein the first cationic polymer is polyethyleneimine (PEI).

8. The fungal drug carrier of any previous claim, wherein the NP is coated with a second cationic polymer.

9. The fungal drug carrier of claim 8, wherein the second cationic polymer is poly-L-lysine.

10. The fungal drug carrier of any previous claim. wherein the NP comprises poly(lactic-co-glycolic) acid (PLGA) or polycaprolactone (PCL). 98Attorney Docket No.:049648-632156 UF Ref. T19361WO001 11. The fungal drug carrier of any one of claims 1-10. wherein the NP is a lipid nanoparticle, liposome, or liposomic formulation.

12. The fungal drug carrier of any one of claims 1-10, wherein the NP comprises an inorganic material.

13. The fungal drug carrier of claim 12, wherein the NP is a gold (Au) NP or an iron oxide (Fe2O3)NP.

14. The fungal drug carrier of any previous claim, wherein the NP is spherical.

15. The fungal drug carrier of any previous claim, wherein the NP has a diameter of about 100 nm to about 500 nm.

16. The fungal drug carrier of claim 15, wherein the NP has a diameter of about 100 nm to about 300 nm.

17. The fungal drug carrier of claim 16, wherein the NP has a diameter of about 200 nm to about 250 nm.

18. The fungal drug carrier of claim 17, wherein the NP has a diameter of about 200 nm.

19. The fungal drug carrier of claim 15, wherein the NP has a diameter of about 400 nm to about 500 nm.

20. The fungal drug carrier of any one of claims 1-10 and 14-19, wherein the NP comprises poly(lactic-co-glycolic) acid (PLGA).

21. The fungal drug carrier of any previous claim, wherein the NP is linked to the surface of the avirulent fungal pathogen cell by electrostatic adsorption.

22. The fungal drug carrier of any one of claims 1-20, wherein the NP is linked to the surface of the avirulent fungal pathogen cell by conjugation. 99Attorney Docket No.:049648-632156 UF Ref. T19361WO001 23. The fungal drug carrier of claim 22, wherein the conjugation of the NP to the avirulent fungal pathogen cell is via a surface amino group on the avirulent fungal pathogen cell.

24. The fungal drug carrier of claim 22, wherein the conjugation of the NP to the avirulent fungal pathogen cell is via a surface thiol group on the avirulent fungal pathogen cell.

25. The fungal drug carrier of any previous claim, wherein the NP is loaded with a small molecule drug or a biologic drug.

26. The fungal drug carrier of any previous claim, wherein the NP is loaded with an anticancer drug, antibiotic drug, or analgesic drug.

27. The fungal drug carrier of claim 25, wherein the NP is loaded with Riluzole or Edaravone.

28. A method of making a fungal drug carrier, wherein the method comprises: (a) providing an avirulent fungal pathogen cell; (b) coating the avirulent fungal pathogen cell with a first cationic polymer; (c) preparing a nanoparticle (NP) comprising a drug; and (d) linking the NP comprising the drug to the surface of the avirulent fungal pathogen coated with the first cationic polymer.

29. A method of making a fungal drug carrier, wherein the method comprises: (a) providing an avirulent fungal pathogen cell; (b) preparing a nanoparticle (NP) comprising a drug and a first cationic polymer; and 100Attorney Docket No.:049648-632156 UF Ref. T19361WO001 (c) linking the NP comprising the drug and the first cationic polymer to the surface of the avirulent fungal pathogen.

30. The method of claim 28 or claim 29, wherein the avirulent fungal pathogen cell is Cryptococcus or Candida albicans.

31. The method of claim 30, wherein the avirulent fungal pathogen cell is Cryptococcus neoformans , Cryptococcus gatti, or Cryptococcus grubii.

32. The method of claim 31, wherein the avirulent fungal pathogen cell is Cryptococcus neoformans.

33. The method of claim 32, wherein the avirulent fungal pathogen cell is Cryptococcus neoformans H99, Cryptococcus neoformans tps1Δ, or Cryptococcus neoformans plb1..

34. The method of any one of claims 28-33, wherein the first cationic polymer is polyethyleneimine (PEI).

35. The method of any one of claims 28-34, wherein the NP comprises poly(lactic-co-glycolic) acid (PLGA) or polycaprolactone (PCL).

36. The method of any one of claims 28-34, wherein the NP is a lipid nanoparticle, liposome, or liposomic formulation.

37. The method of any one of claims 28-34, wherein the NP comprises an inorganic material.

38. The method of claim 37, wherein the NP is a gold (Au) NP or an iron oxide (Fe2O3)NP.

39. The method of any one of claims 28-38, wherein the NP is spherical.

40. The method of any one of claims 28-39, wherein the NP has a diameter of about 100 nm to about 500 nm. 101Attorney Docket No.:049648-632156 UF Ref. T19361WO001 41. The method of claim 40, wherein the NP has a diameter of about 100 nm to about 300 nm.

42. The method of claim 41, wherein the NP has a diameter of about 200 nm to about 250 nm.

43. The method of claim 42, wherein the NP has a diameter of about 200 nm.

44. The method of claim 40, wherein the NP has a diameter of about 400 nm to about 500 nm.

45. The method of any one of 28-35 and 39-44, wherein the NP comprising the drug is a PLGA-NP.

46. The method of claims 28, further comprising a step of coating the NP comprising the drug with a second cationic polymer after step (c).

47. The method of claim 29 further comprising a step of coating the NP comprising the drug and the first cationic polymer with a second cationic polymer after step (b).

48. The method of claim 46 or claim 47, wherein the second cationic polymer is poly-L-lysine.

49. The method of claim 28 or claim 46, wherein the linking of step(d) is by electrostatic adsorption.

50. The method of claim 29 or claim 47, wherein the linking of step(c) is by electrostatic adsorption.

51. The method of claim 28 or claim 46, wherein the linking of step(d) is by conjugation.

52. The method of claim 29 or claim 47, wherein the linking of step(c) is by conjugation. 102Attorney Docket No.:049648-632156 UF Ref. T19361WO001 53. The method of claim 51 or claim 52, wherein the conjugation is via a surface amino group on the avirulent fungal pathogen cell.

54. The method of claim 51 or claim 52, wherein the conjugation is via a surface thiol group on the avirulent fungal pathogen cell.

55. The method of any one of claim 28, 46, 49, 51, 53-55, further comprising a step of functionalizing the PLGA NP comprising the drug before step(d).

56. The method of any one of claims 29, 47, 50, 52, 53, 54, and 56, further comprising a step of functionalizing the PLGA NP comprising the drug before step (c).

57. The method of claim 55 or claim 56, wherein the PLGA NP comprising the drug is functionalized by reaction with EDC and sulfo-NHS to form an NHS-ester- functionalized PLGA NP comprising a drug.

58. The method of any one of claims 45-55, wherein the PLGA NP comprising the drug further comprises PLGA-maleimide.

59. The method of any one of claims 45-55, wherein the PLGA NP comprising the drug further comprises PLGA-NHS.

60. The method of claim 59, wherein the PLGA NP comprising the drug further comprising PLGA-NHS is functionalized by reaction with amine-PEG-azide to form a PLGA-Azide NP comprising a drug.

61. The method of any one of claims 28, 46, 49, 51, 53-55, and 60, further comprising a step of functionalizing the avirulent fungal pathogen cell before step (d).

62. The method of any one of claims 29, 47, 50, 52, 53, 54, 56, and 60, further comprising a step of functionalizing the avirulent fungal pathogen cell before step (c).

63. The method of claim 61 or claim 62, wherein the avirulent fungal pathogen cell is functionalized by reaction with Dibenzocyclooctyne (DBCO). 103Attorney Docket No.:049648-632156 UF Ref. T19361WO001 64. The method of claim 63, wherein the conjugation of step (d) comprises reacting the PLGA-Azide NP comprising the drug with avirulent fungal pathogen cell functionalized by reaction with Dibenzocyclooctyne (DBCO).

65. The method of claim 63, wherein the conjugation of step (c) comprises reacting the PLGA-Azide NP comprising the drug with avirulent fungal pathogen cell functionalized by reaction with Dibenzocyclooctyne (DBCO).

66. The method of any one of claims 28-65, wherein the drug is a small molecule drug or a biologic drug.

67. The method of any one of claims 28-65, wherein the drug is an anticancer drug, antibiotic drug, or analgesic drug.

68. The method of claim 66, wherein the drug is Riluzole or Edaravone.

69. A method of delivering a drug to a central nervous system of a subject, comprising the steps of: (a) providing a fungal drug carrier of any one of claims 1-27; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell across the blood-brain barrier or blood-spinal cord barrier into the central nervous system of the subject, and vomocytosed from the immune cell, delivering the drug to the central nervous system of the subject.

70. The method of claim 69, wherein the drug is delivered to the brain of the subject.

71. The method of claim 69, wherein the drug is delivered to the spinal cord of the subject.

72. A method of delivering a drug to a lymph node of a subject, comprising the steps of: 104Attorney Docket No.:049648-632156 UF Ref. T19361WO001 (a) providing a fungal drug carrier of any one of claims 1-27; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the lymph node of the subject, and vomocytosed from the immune cell, delivering the drug to the lymph node of the subject.

73. A method of delivering a drug to a spleen of a subject, comprising the steps of: (a) providing a fungal drug carrier of any one of claims 1-27; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the spleen of the subject, and vomocytosed from the immune cell, delivering the drug to the spleen of the subject.

74. A method of delivering a drug to a pancreas of a subject, comprising the steps of: (a) providing a fungal drug carrier of any one of claims 1-27; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the pancreas of the subject, and vomocytosed from the immune cell, delivering the drug to the pancreas of the subject.

75. A method of delivering a drug to a lung of a subject, comprising the steps of: (a) providing a fungal drug carrier of any one of claims 1-27; and (b) administering the fungal drug carrier to the subject, 105Attorney Docket No.:049648-632156 UF Ref. T19361WO001 wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the lung of the subject, and vomocytosed from the immune cell, delivering the drug to the lung of the subject.

76. A method of delivering a drug to a heart of a subject, comprising the steps of: (a) providing a fungal drug carrier of any one of claims 1-27; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the heart of the subject, and vomocytosed from the immune cell, delivering the drug to the heart of the subject.

77. A method of delivering a drug to a liver of a subject, comprising the steps of: (a) providing a fungal drug carrier of any one of claims 1-27; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the liver of the subject, and vomocytosed from the immune cell, delivering the drug to the liver of the subject.

78. A method of delivering a drug to a kidney of a subject, comprising the steps of: (a) providing a fungal drug carrier of any one of claims 1-27; and (b) administering the fungal drug carrier to the subject, wherein the fungal drug carrier is engulfed by an immune cell of the subject, transported by the immune cell to the kidney of the subject, and vomocytosed from the immune cell, delivering the drug to the kidney of the subject. 106Attorney Docket No.:049648-632156 UF Ref. T19361WO001 79. The method of any one of claims 69-78, wherein the administering is selected from the group consisting of intranasal, intraperitoneal, intramuscular, intravenous, and subcutaneous.

80. A method of treating a disorder in a subject, the method comprising administering the fungal drug carrier of any one of claims 1-27 to the subject.

81. The method of claim 80, wherein the administering is selected from the group consisting of intranasal, intraperitoneal, intramuscular, intravenous, and subcutaneous.

82. The method of claim 80 or claim 81, wherein the disorder is a central nervous system disorder, a lymph node disorder, a spleen disorder, a pancreatic disorder, a lung disorder, a heart disorder, a liver disorder, a kidney disorder, pancreatic cancer, type 1 diabetes, chronic kidney disease, an infectious disease, an autoimmune disease, a cardiovascular disorder, an angiogenesis-associated disorder, a cancer, or a glycogen storage disease.

83. The method of claim 82, wherein the central nervous system disorder is selected from the group consisting of cancer, epilepsy, stroke, infection, traumatic brain injury, spinal cord injury, a neurodegenerative disorder, amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis.

84. The method of claim 83, wherein the central nervous system disorder is amyotrophic lateral sclerosis.

85. The method of any one of claims 69-84, wherein the subject is a mammal.

86. The method of claim 85, wherein the mammal is a primate.

87. The method of claim 85, wherein the mammal is a non-human primate.

88. The method of claim 85, wherein the mammal is a human.

89. The method of claim 85, wherein the mammal is a rodent.

90. The method of claim 89, wherein the rodent is a mouse, rat, guinea pig, hamster, or gerbil. 107Attorney Docket No.:049648-632156 UF Ref. T19361WO001 91. The method of claim 85, wherein the mammal is selected from the group consisting of human, baboon, chimpanzee, monkey, cynomolgus, marmoset, rhesus, rodent, rabbit, cat, dog, horse, cow, sheep, goat, pig, ferret, guinea pig, hamster, and gerbil.

92. The method of any one of claims 69-84, wherein the subject is a non- human mammal, bird, reptile, amphibian, or fish. 108

Images