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Title:
FUNGAL NANOPARTICLES AND METHODS OF MAKING AND USING THEREOF
Document Type and Number:
WIPO Patent Application WO/2014/066509
Kind Code:
A1
Abstract:
Nanoparticles produced by and isolated from a fungus of the genus Arthrobotrys are disclosed and can comprise at least about 1% by weight of a glycosaminoglycan. Also disclosed are fractions obtained from the nanoparticles. Methods of preparing and isolating these nanoparticles and fractions are also disclosed. The disclosed nanoparticles and fractions are suitable as carries in a variety of medical formulations. Thus, in a further aspect, the present disclosure relates to compositions comprising the disclosed nanoparticles and fractions with carriers, excipients, and/or therapeutic agents. Methods of treating subjects with these compositions are also disclosed.

Inventors:
ZHANG MINGJUN (US)
WANG YONGZHONG (US)
Application Number:
PCT/US2013/066393
Publication Date:
May 01, 2014
Filing Date:
October 23, 2013
Export Citation:
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Assignee:
ZHANG MINGJUN (US)
WANG YONGZHONG (US)
International Classes:
C12N1/14; A61K35/66; A61P35/00
Foreign References:
US20100055199A12010-03-04
US20120164062A12012-06-28
Other References:
DHILLON, GURPREET SINGH ET AL.: "Green approach for nanoparticle biosynthesis by fungi: current trends and applications", CRITICAL REVIEWS IN BIOTECHNOLOGY, vol. 32, no. 1, 22 June 2011 (2011-06-22), pages 49 - 73
AHMAD, ABSAR ET AL.: "Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum", COLLOIDS AND SURFACES B: BIOINTERFACES, vol. 28, no. 4, 1 May 2003 (2003-05-01), pages 313 - 318
YEN SAN, CHAN ET AL.: "Instantaneous biosynthesis of silver nanoparticles by selected macro fungi", AUSTRALIAN JOURNAL OF BASIC AND APPLIED SCIENCES, vol. 6, no. 1, January 2012 (2012-01-01), pages 222 - 226
WANG, YONGZHONG ET AL.: "Naturally Occurring Nanoparticles from Arthrobotrys oligospora as a Potential Immunostimulatory and Antitumor Agent", ADVANCED FUNCTIONAL MATERIALS, vol. 23, no. 17, 4 December 2012 (2012-12-04), pages 2175 - 2184
ZHANG, MINGJUN: "Fungus-based nanoparticles: inspiration from nature for cancer therapy", NANOMEDICINE, vol. 8, no. 3, March 2013 (2013-03-01), pages 313 - 316
Attorney, Agent or Firm:
CURFMAN, Christopher L. et al. (LLCSuite 500,817 W. Peachtree Street N, Atlanta Georgia, US)
Download PDF:
Claims:
CLAIMS

What is claimed is:

1. Isolated nanoparticles, comprising at least about 1% by weight of a

glycosaminoglycan, wherein the nanoparticles are produced by a fungus of the genus Arthrobotrys .

2. The nanoparticles of claim 1, wherein the nanoparticles are produced by

Arthrobotrys oligospora.

3. The nanoparticles of any one of the preceding claims, wherein the average diameter of the nanoparticles are from about 50 nm to about 800 nm.

4. The nanoparticles of any one of the preceding claims, wherein the average diameter of the nanoparticles are from about 100 nm to about 500 nm.

5. The nanoparticles of any one of the preceding claims, wherein the

nanoparticles have a zeta potential of from about -10 mV to about -50 mV at about pH 6.0.

6. The nanoparticles of any one of the preceding claims, wherein the

nanoparticles comprise at least about 30 μg of glycosaminoglycan per 1 mg of nanoparticles.

7. The nanoparticles of any one of the preceding claims, wherein the percentage of glycosaminoglycan in the nanoparticles is at least about 15% (w/w).

8. The nanoparticles of any one of the preceding claims, wherein the

nanoparticles comprise a first fraction that elutes with relatively lower salt content buffer and a second fraction that elutes with relatively higher salt content buffer.

9. The nanoparticles of claim 8, wherein the percentage of glycosaminoglycan in the first fraction is at least about 25% (w/w).

10. The nanoparticles of claim 8, wherein the percentage of glycosaminoglycan in the second fraction is at least about 60% (w/w).

11. Isolated nanoparticles, comprising at least 25% (w/w) of glycosaminoglycan, wherein the nanoparticles are fractions of original nanoparticles produced by a fungus of the genus Arthrobotrys .

12. The nanoparticles of claim 11 , wherein the average diameter of the

nanoparticles is from about 50 nm to about 300 nm.

13. The nanoparticles of any one of claims 11-12, wherein the average diameter of the nanoparticles is from about 100 nm to about 200 nm.

14. The nanoparticles of any one of claims 11-13, having a zeta potential of from about -25 mV to about -40 mV at about pH 6.0.

15. The nanoparticle of any one of claims 11-14, wherein the percentage of

glycosaminoglycan in the original nanoparticles is a least about 15% (w/w/).

16. The nanoparticles of any one of claims 11-15, wherein the nanoparticles are a first fraction that elutes with relatively lower salt content buffer or a second fraction that elutes with relatively higher salt content buffer from the original nanoparticles.

17. The nanoparticles of claim 16, wherein the nanoparticles from the first fraction has a zeta potential lower than that of the nanoparticles from the second fraction.

18. The nanoparticles of claim 16, wherein the nanoparticles from the first fraction has the percentage of glycoaminoglycan higher than that of the nanoparticles from the second fraction.

19. The nanoparticles of any one of claims 1 1-18, having the percentage of

glycosaminoglycan at least about 35% (w/w/).

20. The nanoparticles of any one of claims 1 1-19, having the percentage of glycosaminoglycan at least about 80% (w/w).

21. The nanoparticles of any one of claims 1-20, further comprising a therapeutic agent linked to or complexed with the nanoparticles.

22. The nanoparticle of claim 21, wherein the therapeutic agent is Doxorubicin, Indarubicin, 9-aminocamptothecin, or Exatecan.

23. A method of reducing tumor growth and/or inducing immunostimulation in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of nanoparticles of any one of claims 1-22.

24. The method of claim 23, wherein the nanoparticles produce

immunostimulation in the subject.

25. The method of claim 23, wherein the nanoparticles arrest growth of tumor cells in the subject.

26. The method of any one of claims 22-25, wherein the nanoparticles produce immunostimulation and arrest growth of tumor cells in the subject, and the immunostimulation and arresting effects are synergistic.

27. The method of any one of claims 22-26, wherein composition further

comprises an anticancer therapeutic.

28. A method of producing a plurality of nanoparticles, the method comprising, culturing a fungus of the genus Arthrobotrys to develop filamentous mycelia having fungal secreted nanoparticles associated with the mycelia, and separating the nanoparticles from the mycelia to form the plurality of nanoparticles.

29. The method of claim 28, wherein the fungus is Arthrobotrys oligospora.

30. The method of any one of claims 28-29, wherein the separating is conducted by washing the fungal mycelia with distilled water followed by filtration to remove the debris and collecting the nanoparticles as filtrate.

31. The method of any one of claims 28-30, wherein the separating is conducted by sonicating the fungal mycelia in distilled water followed by filtration to remove the debris and collecting the nanoparticles as filtrate.

32. The method of any one of claims 28-31 , further comprising removing small organic molecules from the nanoparticles through dialysis.

33. The method of any one of claims 28-32, wherein the plurality of nanoparticles each comprising at least about 1% by weight a glycosaminoglycan

composition based on the total chemical composition of the nanoparticle,

34. The method of any one of claims 28-33, wherein the average diameter of the nanoparticles is from about 50 nm to about 800 nm.

35. The method of any one of claims 28-34, wherein the nanoparticles each have a zeta potential of from about -10 mV to about -50 mV at about pH 6.0.

36. The method of any one of claims 28-35, further comprising desalting the

nanoparticles using an ion exchange medium.

37. The method of any one of claims 28-36, further comprising fractionating the nanoparticles with weak anion-exchange chromatography using a salt gradient.

38. The method of claim 37, wherein the salt gradient is a stepwise gradient and the fractionation produces a first fraction that elutes with relatively a lower salt content buffer and a second fraction that elutes with a relatively higher salt content buffer and the method further comprising forming nanoparticles from first fraction and forming nanoparticles from second fraction.

39. The method of claim 38, wherein the first fraction has a percentage of

glycosaminoglycan of at least about 15% (w/w). The method of claim 38, wherein the first fraction has a percentage of glycosaminoglycan of at least about 80% (w/w).

The method of any one of claims 37-40, wherein the average diameter of the nanoparticles formed from the fractions is from about 50 nm to about 300 nm.

The method of any one of claims 37-41, wherein the nanoparticles formed from the fractions each has a zeta potential of from about -25 mV to about -40 mV at about pH 6.0.

The method of any one of claims 28-42, further comprising covalently linking or non-covalently complexing a therapeutic agent with the nanoparticles.

The method of claim 43, further comprising covalently linking or non- covalently complexing doxorubicin with the nanoparticles.

Description:
FUNGAL NANOPARTICLES AND METHODS OF MAKING AND

USING THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional

Application No. 61/717,433, filed on October 23, 2012, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The present disclosure was made with Government Support under Grant No. ARO W91 lNF-10-1-0114 awarded by the U.S. Army Research Office. The Government has certain rights in this disclosure.

TECHNICAL FIELD

[0003] The subject matter disclosed herein generally relates to organic nanoparticles produced from fungus under controlled conditions and methods of making and using these nanoparticles and compositions and conjugates comprising these nanoparticles.

BACKGROUND

[0004] Cancer is a leading cause of death worldwide, accounting for about 7.6 million deaths (around 13% of all deaths) in 2008, and the deaths are expected to continue rising, with an estimated 13.1 million in 2030. Chemotherapy is generally regarded as the first line approach for the treatment of malignant cancer in the past decades. However, conventional chemotherapy remains a daunting challenge to the successful treatment of metastatic tumors. It becomes ineffective in many patients after the first few treatments, which has been attributed to tumor heterogeneity, plasticity, and ineffective drug delivery to tumor tissues and cells that allow a subgroup of cancer cells to mutate and evade the chemotherapy. Simply increasing drug dose usually does not guarantee elimination of this subset of tumor cells, but could eventually lead to systemic toxicity in normal tissues and high frequency multidrug resistance in tumor cells.

[0005] To evade the emergence of systemic toxicity and therapy resistance, the development of new treatment modalities with multiple mechanisms of cell killing in tumors is needed. One option is combined immunochemotherapy, which has demonstrated great potential in maximizing the clinical outcomes of cancer patients due to its synergistic antitumor effects between chemotherapy and immunotherapy. Various clinical trials incorporating cancer vaccines, immune checkpoint blockade, or adoptive cellular therapy have tested immunotherapies integrated with standard-dose chemotherapy. Current clinical data suggests that combined immunochemotherapies are not only drug dependent, but also dependent on drug dose, timing and schedule related immune -based intervention. Because the strategy conditionally depends on synergism between chemotherapy and immunotherapy, developing rational carriers that could incorporate and simultaneously deliver both immune-stimulating and cytotoxic chemotherapeutic agents can facilitate better temporal and spatial delivery of different therapeutics. From this perspective, a few combination therapies using engineered nanoparticle-based delivery systems, including nanoparticles, liposomes and macromolecular conjugates in conjunction with different chemical drugs and immune-stimulants, have been reported. However, among these nanoparticle- enhanced combination immunochemotherapies, few engineered biomaterials play the role of immunostimulants or adjuvants. They are usually inert biomaterials, simply conjugated or encapsulated with an immunostimulatory agent and a chemo-drug in the combined antitumor therapy. Due to the complex pathogenesis of malignancy, which juxtaposes intrinsic aberrations in tumor cells with profound effects on the host innate and acquired immune systems, a multi-functional therapeutic strategy that can target tumor cells and improve antitumor immune responses is highly desired.

Immunochemotherapies call for more novel therapeutic biomaterials with multifunctional modes of action, in conjunction with conventional drugs. The

compositions and methods disclosed herein address these and other needs.

SUMMARY

[0006] The subject matter disclosed herein relates to compositions and methods of making and using the compositions. The present disclosure relates to organic nanoparticles and methods of making and using thereof. The present disclosure additionally relates to nanoparticles produced by and isolated from a fungus of the genus Arthrobotrys . The disclosed nanoparticles can comprise at least about 1% by weight of a glycosaminoglycan. Also disclosed are fractions obtained from the nanoparticles. Methods of preparing and isolating these nanoparticles and fractions are also disclosed. The disclosed nanoparticles and fractions are suitable as carries in a variety of medical formulations. Thus, in a further aspect, the present disclosure relates to compositions comprising the disclosed nanoparticles and fractions with carriers, excipients, and/or therapeutic agents. Methods of treating subjects with these compositions are also disclosed.

[0007] Additional advantages of the disclosed subject matter will be set forth in part in the description that follows, and in part will be obvious from the description, or can be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE FIGURES

[0008] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0009] FIG. 1 is a schematic showing the fungal sitting drop culture method. Briefly, a drop of medium was added onto a sterile cover slip and placed into a small Petri dish (3 cm), and then 50-100 conidia of A. oligospora from a stock culture on CM A dish were inoculated into the drop. Humidity was maintained by filling another small Petri dish with water, and placing both small Petri dish into a large one (10 cm). The low nutrient medium (LNM) supplemented with 1 mg/ml phe-val was used as a liquid drop for the fungal growth. The drop culture device was incubated at 25°C for 15 days until the nanoparticles were harvested.

[0010] FIG. 2 A shows inverted optical microscopy of the A. oligospora growth in a medium drop on the cover slip on day 5 post-inoculation. FIG. 2B shows inverted optical microscopy of A. oligospora mycelia with 3D-traps on the cover slip on day 5 post-inoculation.

[0011] FIG. 3 shows the SEM/AFM images of nanoparticles (naturally occurring nanoparticles) generated from A. oligospora cultured in the drop culture system.

[0012] FIG . 4 shows Fourier transform infrared (FTIR) spectra of ( 1 ) NONP- W, (2) NONP-DOX conjugates and (3) free DOX. [0013] FIG. 5 shows effects of nanoparticles from A. oligospora on the production of TNF-a, and IL-12 in RAW 264.7 macrophage cells.

[0014] FIG. 6 shows in vitro cytotoxicity of the nanoparticles or the NONP-DOX conjugates against human non-small-cell lung cancer A549 cells (A and C) and mouse melanoma B16BL6 cells (B and D).

[0015] FIG. 7 shows flow cytometry analysis for cellular uptake of NONP-DOX conjugates in human lung cancer A549 cells (A) and mouse melanoma B16BL6 cells (B); and statistical differences between DOX and NONP-DOX conjugates observed for respective cell line were compared (C).

[0016] FIG. 8 shows intracellular distributions of NONP-DOX conjugates and free DOX at the DOX concentration of 10 μΜ in human lung cancer A549 cells mouse (A) and mouse melanoma B16BL6 cells (B).

[0017] FIG. 9A shows the elution profile of FNP0. FIG. 9B shows the elution profiles of FNP1 and FNP2.

[0018] FIGs. 10A-C shows AFM images (A-B) and size distributions (C) of FNP0. FIGs. 10D-F shows AFM images (D-E) and size distributions (F) of FNP1. FIGs. 10G-I shows AFM images (G-H) and size distributions (I) of FNP2.

[0019] FIG. 11 shows SDS-PAGE analysis of polysaccharides in the FNPs.

[0020] FIG. 12 shows the effects of the FNPs on the secretion of cytokines (A and B), chemokines (C and D) and nitric oxide (E and F) from RAW 264.7 macrophage cells (A, C and E) and splenocytes (B, D and F).

[0021] FIG. 13 shows characterization of the DOX-FNP complexes (A-D) and pH-responsive release of DOX from the complexes (E).

[0022] FIG. 14 shows cytotoxicity of the FNPs and the DOX-FNPs complexes against multiple cell lines.

[0023] FIG. 15 shows apoptosis (A-B) and cell cycle arrest (C-D) in human non- small-cell lung cancer A549 cells (A and C) and mouse melanoma B16BL6 cells (B and D) induced by the FNPs.

[0024] FIG. 16 shows the results from quantitative analyses of DOX uptake by human non-small-cell lung cancer A549 cells (A-B) and mouse melanoma B16BL6 cells (C-D). [0025] FIG. 17 shows confocal images of intracellular distribution of the DOX-

FNP complexes at the DOX concentration of 10 μΜ in human non-small-cell lung cancer A549 cells (A) and mouse melanoma B16BL6 cells (B).

[0026] FIG. 18 shows chemo-immunotherapeutic activities of the DOX-FNP complexes in an in vitro experimental system where B16BL6 tumor cells were first labeled with CFSE and then co-cultured with the splenocytes derived from C57BL/6 mice.

[0027] FIG. 19 shows a mechanism of NONP-doxorubicin conjugation.

DETAILED DESCRIPTION

[0028] The compositions and methods described herein can be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples and Figures included therein.

[0029] Before the present compositions and methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0030] Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

General Definitions

[0031] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0032] As used in the description and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "the nanoparticle" includes mixtures of two or more such nanoparticles, and the like. [0033] "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0034] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a mixture containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the mixture.

[0035] A weight percent (wt.%) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0036] A "pharmaceutically acceptable" component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

[0037] "Pharmaceutically acceptable salt" refers to a salt that is pharmaceutically acceptable and has the desired pharmacological properties. Such salts include those that may be formed where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g., sodium, potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids {e.g., hydrochloric and hydrobromic acids) and organic acids {e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). When two acidic groups are present, a pharmaceutically acceptable salt may be a mono-acid-mono-salt or a di-salt;

similarly, where there are more than two acidic groups present, some or all of such groups can be converted into salts.

[0038] "Pharmaceutically acceptable excipient" refers to an excipient that is conventionally useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0039] A "pharmaceutically acceptable carrier" is a carrier, such as a solvent, suspending agent or vehicle, for delivering the disclosed compounds to the patient. The carrier can be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutical carrier. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

[0040] The term "therapeutically effective amount" as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. In reference to cancers or other unwanted cell proliferation, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some examples, an effective amount is an amount sufficient to delay development. In some examples, an effective amount is an amount sufficient to prevent or delay occurrence and/or recurrence. An effective amount can be administered in one or more doses. In the case of cancer, the effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

[0041] Effective amounts of a compound or composition described herein for treating a mammalian subject can include about 0.1 to about 1000 mg/Kg of body weight of the subject/day, such as from about 1 to about 100 mg/Kg/day, especially from about 10 to about 100 mg/Kg/day. The doses can be acute or chronic. A broad range of disclosed composition dosages are believed to be both safe and effective.

[0042] Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0043] As used herein, by a "subject" is meant an individual. Thus, the "subject" can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. "Subject" can also include a mammal, such as a primate or a human.

[0044] By "reduce" or other forms of the word, such as "reducing" or "reduction," is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, "reduces tumor growth" means reducing the rate of growth of a tumor relative to a standard or a control.

[0045] By "prevent" or other forms of the word, such as "preventing" or

"prevention," is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0046] By "treat" or other forms of the word, such as "treated" or "treatment," is meant to administer a composition or to perform a method in order to reduce, prevent, inhibit, or eliminate a particular characteristic or event (e.g. , tumor growth or survival). The term "control" is used synonymously with the term "treat."

[0047] Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.

Nanoparticles

[0048] Naturally occurring organic nanoparticles have drawn significant interest from scientific communities due to their unique properties and increased

biocompatibility. It was discovered that ivy secreted organic nanoparticles, and that these nanoparticles aided in the generation of the strong adhesive force that allows ivy to climb vertical surfaces (Zhang et ah, Nano Lett 2008;8(5): 1277-80; Lenaghan and Zhang, Plant Sci 2012;183:206-11). Further study found that the ivy nanoparticles were less toxic than similarly sized Ti0 2 and ZnO nanoparticles, which makes them an attractive candidate for UV fillers in sunscreens (Xia et al., J Nanobiotech

2010;8(1): 12). Similar organic nanostructures have been discovered in the secretions of a variety of marine species, including polychaetes, mussels, barnacles, and sea stars. In fact, low-density lipoprotein (LDL) present in mammalian blood has long been recognized as a naturally occurring nanoparticle. LDL serves as the main transport vehicle for cholesterol in mammalian systems, and bio-inspired LDLs have been used as potential carriers for targeted delivery of diagnostic and therapeutic agents.

[0049] Most studies on naturally occurring organic nanoparticles have focused on higher organisms. However, disclosed herein are nanoparticles that are produced by and isolated from various fungi.

[0050] The nanoparticles disclosed herein are secreted from certain fungi and have an average diameter of from about 50 nm to about 1000 nm (e.g. from about 50 nm to about 800 nm, from about 75 nm to about 900 nm, from about 100 nm to about 800 nm, from about 125 nm to about 700 nm, from about 150 nm to about 600 nm, from about 100 nm to about 500 nm, or from about 300 nm to about 400 nm). In some examples, the nanoparticles have an average diameter of from about 360 nm to about 370 nm. The disclosed nanoparticles can have a zeta potential of from about - 10 mV to about -50 mV at pH 6.0. (e.g. from about -15 mV to about -45 mV, from about -20 mV to about -40 mV, or from about -25 mV to about -35 mV). In some examples, the disclosed nanoparticles have a zeta potential of about -33 mV at pH 6.0. Each of the nanoparticles disclosed herein can comprise at least about 10 μ (e.g. at least about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, or about 100 μg, where any of the stated values can form an upper or lower endpoint of a range) of the glycosaminoglycan per 1 mg of the nanoparticle. Each of the nanoparticles disclosed herein can have glycosaminoglycan that is at least about 5% based on molar percentage or mol/mol% (e.g. at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%), or about 15%>, where any of the stated values can form an upper or lower endpoint of a range). The disclosed nanoparticles can have immunostimulatory effect through inducing tumor necrotic factor (TNF-a) secretion in animal macrophages. The disclosed nanoparticles can have mild cytotoxicity to tumor cells and showed synergistic cytotoxic effect upon conjugation with doxorubicin against tumor cells.

[0051] The disclosed nanoparticles can be obtained from Arthrobotrys, which is a genus of mitosporic fungi in the family Orbiliaceae. They are predatory fungi that capture and feed on roundworms. Rings or traps that form on the hyphae constrict and entrap the worms; then filamentous mycelia such as hyphae grow into the worm and digest it. There are 71 species in the Arthrobotrys genus, including Arthrobotrys aggregate, Arthrobotrys alaskana, Arthrobotrys amerospora, Arthrobotrys anomala, Arthrobotrys apscheronica, Arthrobotrys arthrobotryoides, Arthrobotrys

azerbaijanica, Arthrobotrys bakunika, Arthrobotrys botryospora, Arthrobotrys brochopaga, Arthrobotrys chazarica, Arthrobotrys chilensis, Arthrobotrys cladodes, Arthrobotrys clavispora, Arthrobotrys compacta, Arthrobotrys conoides, Arthrobotrys constringens, Arthrobotrys cylindrospora, Arthrobotrys dactyloides, Arthrobotrys deflectens, Arthrobotrys dendroides, Arthrobotrys doliiformis, Arthrobotrys drechsleri, Arthrobotrys elegans, Arthrobotrys ellipsospora, Arthrobotrys

entomopaga, Arthrobotrys ferox, Arthrobotrys foliicola, Arthrobotrys fruticulosa, Arthrobotrys globospora, Arthrobotrys haptospora, Arthrobotrys hertziana,

Arthrobotrys indica, Arthrobotrys irregularis, Arthrobotrys javanica, Arthrobotrys kirghizica, Arthrobotrys longa, Arthrobotrys longiphora, Arthrobotrys longiramulifera, Arthrobotrys longispora, Arthrobotrys mangrovispora, Arthrobotrys megaspore, Arthrobotrys microscaphoides, Arthrobotrys microspora, Arthrobotrys multisecundaria, Arthrobotrys musiformis, Arthrobotrys nematopaga, Arthrobotrys nonseptata, Arthrobotrys oligospora, Arthrobotrys oudemansii, Arthrobotrys oviformis, Arthrobotrys perpasta, Arthrobotrys polycephala, Arthrobotrys

pseudoclavata, Arthrobotrys pyriformis, Arthrobotrys recta, Arthrobotrys robusta, Arthrobotrys rosea, Arthrobotrys scaphoides, Arthrobotrys sclerohypha, Arthrobotrys shahriari, Arthrobotrys shizishanna, Arthrobotrys sinensis, Arthrobotrys soprunovii, Arthrobotrys stilbacea, Arthrobotrys straminicola, Arthrobotrys superb, Arthrobotrys tabrizica, Arthrobotrys venusta, Arthrobotrys vermicola, and Arthrobotrys yunnanensis. Any of these species, or any combination of these species, can be used to prepare the disclosed nanoparticles or fractioned nanoparticles (FNP) suitable for use herein.

[0052] For example, Arthrobotrys oligospora in particular has specialized 3D adhesive traps that can capture, penetrate and digest free-living nematodes in diverse environments. A. oligospora, a representative flesh eater with a saprophytic and predatory life stage is disclosed herein as a representative fungal species that can be used to obtain the disclosed nanoparticles. In the presence of nematodes or proteinaceous substances, A. oligospora can change from a saprophyte into a predatory stage, characterized by the formation of 3D adhesive trapping networks that can trap nematodes for subsequent digestion. The broad adaptability and flexible lifestyle of the fungus makes it an attractive candidate for the control of parasitic nematodes in both plants and animals. Several biopolymers from the 3D-trapping networks of A. oligospora have been reported, and it is believed that both proteins and carbohydrates are involved in the adhesion process.

[0053] As disclosed herein, nanoparticles that are produced by fungi from the genus Arthrobotrys or other similar fungi are isolated, purified, and optionally fractionated and used in biomedical applications and compositions. A fungal sitting drop culture method can be used to produce the nanoparticles under controlled conditions. The sitting drop culture method disclosed herein can also be used to monitor the growth of fungi in situ and observe nanoparticle production without interfering or contamination from the solid media. [0054] Also disclosed herein are purification or fractionation procedures for obtaining nanoparticles and that produce desalted fraction nanoparticles "FNP0" through a size exclusion media. The FNP0 can be further fractionated on a weak anion exchange media to produce a first fraction nanoparticles "FNPl" eluted by a lower salt content buffer (i.e., 0.5 M NaCl) and a second fraction nanoparticles "FNP2" eluted by a higher salt content buffer (i.e., 1.0 M NaCl). Polysaccharides, including glycosaminoglycan, are the main constituents in the various fractioned nanoparticles "FNPs." "FNPs" is used herein to refer to either FNP0, FNPl, or FNP2 individually or collectively, unless the context dictates otherwise.

[0055] The fraction FNP0 can have an average diameter from about 50 nm to about 1000 nm (e.g. from about 50 to about 800 nm, from about 75 nm to about 900 nm, from about 100 nm to about 800 nm, from about 125 nm to about 700 nm, from about 150 nm to about 600 nm, from about 100 nm to about 500 nm, or from about 300 nm to about 400 nm). In some examples, the fraction FNP0 can have an average diameter of about 300 nm. The fraction FNP0 can haves a zeta potential of from about -25 mV to about -40 mV at about pH 6.0. (e.g. from about -26 mV to about -38 mV, from about -27 mV to about -36 mV, or from about -28 mV to about -34 mV). In some examples, the fraction FNP0 can have a zeta potential of about -30 mV at pH 6.0.

[0056] Similarly, the disclosed fractions FNPl and FNP2 can have an average diameter of from about 50 nm to about 500 nm (e.g. from about 75 nm to about 450 nm or from about 200 nm to about 400 nm). In some examples, the fractions FNPl or FNP2 can have an average diameter of about 300 nm. The fractions FNPl or FNP2 can have a zeta potential of from about -25 mV to about -40 mV at about pH 6.0. (e.g., from about -26 mV to about -38 mV, from about -27 mV to about -36 mV, or from about -28 mV to about -34 mV). In some examples, the fractions FNPl and FNP2 can have a zeta potential of about -30 mV at pH 6.0.

[0057] In some examples, the FNPs can have a zeta potential of about -33 mV at pH 6.0. The disclosed nanoparticles or FNPs can comprise at least about 10 μg (e.g., at least about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, or about 100 μg, where any of the stated values can form an upper or lower endpoint of a range) of glycosaminoglycan per 1 mg of the nanoparticle. The disclosed nanoparticles or FNPs can have glycosaminoglycan in the nanoparticle that is at least about 5% based on molar percentage or mol/mol% (e.g. at least about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%), about 14%, or about 15%). The disclosed nanoparticles or FNPs can have a percentage of glycosaminoglycan in of at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, or about 85% (w/w).

[0058] The FNPs are demonstrated herein to enhance the secretion of multiple proinflammatory cytokines and chemokines from macrophages and splenocytes, indicating the efficacy of the FNPs as immunomodulators of biological responses in the adjuvant antitumor therapy. The unfractionated nanoparticles can be similarly used. MTT assay shows that the two purified FNPs had mild cytotoxicity against multiple tumor cells, but the FNP2 had stronger cytotoxic activity than the FNP1. The differences in the cytotoxic activity between the two FNPs were substantiated by apoptosis and cell cycle analysis. FNP2 could clearly inhibit cell proliferation via inducing apoptosis and arresting tumor cells at sub G0/G1 phase (FIG. 15). Both FNP1 and FNP2 are shown herein to form pH-responsive nanocomplexes with doxorubicin (DOX) via electrostatic interactions. In a direct cytotoxicity experiment, the DOX-FNP2 complexes demonstrated higher cytotoxic activity than the free DOX against multiple tumor cells, while the cytotoxic activity of the DOX-FNP1 complexes was weaker than the free DOX. Interestingly, in a co-culture experiment where splenocytes were co-cultured with tumor cells, both DOX-FNP complexes demonstrated higher antitumor activity than the free DOX, indicating a synergistic effect between the immunostimulation of the FNPs and cytotoxicity of the

nanocomplexes in vitro.

[0059] The disclosed nanoparticles and FNPs are substantially pure. By substantially pure is meant that the nanoparticles are free from cell debris and solvents

(e.g., less than about 1% by weight is debris or solvent).

Compositions, Formulations and Methods of Administration

[0060] The disclosed nanoparticles and FNPs can be used in compositions along with a variety of anti-cancer therapeutics for various treatments. In this way the disclosed nanoparticles or FNPs can act as multifunctional nanocarriers. Such compositions can be used for in vivo application by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed nanoparticles or FNPs can be formulated alone or with other

therapeutics in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. Thus disclosed herein are compositions that comprise nanoparticles obtained from fungi or fractions thereof, as disclosed herein, and a pharmaceutically acceptable excipient or carrier, and optionally an anticancer therapeutic. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. Administration of the disclosed compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art.

[0061] The disclosed compositions can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington 's Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the disclosed nanoparticles and FNPs can be formulated such that an effective amount is combined with a suitable carrier and/or with a suitable anti-cancer therapeutic in order to facilitate effective administration of the compound. The disclosed compositions can also be used in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99%, and especially, 1 and 15% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

[0062] Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question.

[0063] For the treatment of cancer, the disclosed nanoparticles or FNPs can be administered to a subject in need of treatment in combination with other antitumor or anticancer substances and/or with radiation and/or photodynamic therapy and/or with surgical treatment to remove a tumor. These other substances or treatments can be given at the same as or at different times from the compositions disclosed herein. For example, the disclosed nanoparticles or FNPs can be used in combination with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and/or other anti-cancer drugs or antibodies, such as, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.), respectively. Further examples of some chemotherapeutic agents that can be used with or attached to the disclosed nanoparticles or FNPs include 13-cis-Retinoic Acid, 2-Amino-6-Mercaptopurine, 2-CdA, 2-Chlorodeoxyadenosine, 5-fluorouracil, 6-Thioguanine, 6-Mercaptopurine, Accutane, Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alitretinoin, Alkaban-AQ, Alkeran, All-transretinoic acid, Alpha interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron, Anastrozole, Arabinosylcytosine, Aranesp, Aredia, Arimidex, Aromasin, Arsenic trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib, Busulfan, Busulfex, C225, Calcium Leucovorin, Campath, Camptosar, Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine, Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cerubidine, cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen, CPT- 11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine liposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposomal, DaunoXome, Decadron, Delta- Cortef, Deltasone, Denileukin diftitox, DepoCyt, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposomal, Droxia, DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt,

Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate, Eulexin, Evista, Exemestane, Fareston, Faslodex, Femara, Filgrastim, Floxuridine, Fludara, Fludarabine, Fluoroplex,

Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron, Lupron Depot, Matulane, Maxidex, Mechlorethamine, - Mechlorethamine Hydrochlorine, Medralone, Medrol, Megace, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate Sodium, Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta, Neumega, Neupogen, Nilandron, Nilutamide, Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide acetate, Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orapred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin,

Pediapred, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L- asparaginase, Phenylalanine Mustard, Platinol, Platinol-AQ, Prednisolone,

Prednisone, Prelone, Procarbazine, PROCRIT, Proleukin, Prolifeprospan 20 with Carmustine implant, Purinethol, Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alfa-2a), Rubex, Rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin, Taxol, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide, Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab, Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid, Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Vumon, Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zometa, Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte colony stimulating factor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, HMM, Hycamtin, Hydrea, Hydrocort Acetate, Hydrocortisone, Hydrocortisone sodium phosphate, Hydrocortisone sodium succinate, Hydrocortone phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin, Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib mesylate, Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEG conjugate),

Interleukin 2, Interleukin-11, Intron A (interferon alfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine, Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, L-PAM, L-Sarcolysin, Meticorten, Mitomycin, Mitomycin-C,

Mitoxantrone, M-Prednisol, MTC, MTX, Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan, Isotretinoin, Kidrolase, Lanacort, L-asparaginase, and LCR. In a preferred example the disclosed nanoparticles or FNPs are used with or attached to dioxyrubicin. Particularly preferred therapeutics that can be combined or conjugated to the disclosed nanoparticles or FNPs include Doxorubicin, Indarubicin, 9- aminocamptothecin, and Exatecan.

[0064] The compositions disclosed herein can be locally administered at one or more anatomical sites, such as sites of unwanted cell growth (such as a tumor site or benign skin growth, e.g., injected or topically applied to the tumor or skin growth), optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. The compositions disclosed herein can be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent, or an assimilable edible carrier for oral delivery. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the compositions can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

[0065] The tablets, troches, pills, capsules, and the like can also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring can be added. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir can contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as

preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound can be incorporated into sustained-release preparations and devices.

[0066] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.

Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0067] Sterile injectable solutions are prepared by incorporating a compound and/or agent disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0068] For topical administration, the compositions disclosed herein can be applied in as a liquid or solid. However, it will generally be desirable to administer them topically to the skin as compositions, in combination with a dermatologically acceptable carrier, which can be a solid or a liquid. Compositions disclosed herein can be applied topically to a subject's skin to reduce the size (and can include complete removal) of malignant or benign growths, or to treat an infection site. Compositions disclosed herein can be applied directly to the growth or infection site. Preferably, the compositions are applied to the growth or infection site in a formulation such as an ointment, cream, lotion, solution, tincture, or the like.

[0069] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example.

[0070] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0071] As noted herein the disclosed nanoparticles and FNPs can act as nanocarriers such that these nanoparticles are administered with a therapeutic. The therapeutic can associate with the nanoparticles by noncovalent bonding (e.g., electrostatic interactions). In particular, any therapeutic (e.g., anticancer drug) with a positive charge at neutral pH can be use with, and thus carried by, the disclosed nanoparticles or FNPs. It is also contemplated that the disclosed nanoparticles and FNPs can be conjugated to a therapeutic whereby the therapeutic is bonded to the nanoparticle by a covalent bond. An example of conjugation is shown in FIG. 19. In several examples, the disclosed nanoparticles or FNPs contain carboxylic groups that can be coupled to reactive groups, such as hydroxyl, amine, or carbonyl groups, on a therapeutic molecule. One suitable way to effect such a coupling reaction is to use a carbodiimide-mediated coupling to form a bond between the therapeutic and the nanoparticles. For example, a therapeutic with a hydroxyl or amine group can be coupled to the nanoparticle with carboxylate or carboxylic acid functional groups using carbodiimides such as l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1 -cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-tolue ne sulfonate, and N,N'-dicyclohexylcarbodiimide.

Abundant mycelia and 3D-traps development using a fungal sitting drop culture system

[0072] The FNPs collected by a washing-dialysis procedure from a previous study showed a size of from about 200 to about 300 nm in diameter measured by

SEM/AFM, and from about 300 to about 400 nm in aqueous solution measured by DLS. From the perspective of passive tumor targeting in vivo, the upper bound size of the nanoparticles participating in the enhanced permeability and retention (EPR) effect is believed to be about 400 nm, and an effective drug carrier for in vivo cancer treatment should have a diameter less than about 200 nm considering the multiple factors in vivo, such as limited fenestration size of the leaky vasculature in tumors and rapid systemic clearance. Thus, a method for purification of the crude FNPs from the sitting drop culture system was established, followed by characterization of physical- chemical properties of the purified FNPs. The purified FNPs were demonstrated as a multifunctional nanocarrier in cancer immune-chemotherapy by investigating immunostimulatory activities in macrophages and splenocytes, cytotoxicities, apoptotic effects, cell cycle arrests and delivery of chemo-drug in different tumor cells, as well as the combined immune-chemotherapeutic effects in an in vitro co- culture system.

[0073] A culture method, fungal sitting drop culture method, was established in order to monitor the growth of Arthrobotrys species in situ and observe the nanoparticle production without interfering or contamination from the solid media. Abundant spherical nanoparticles secreted from the fungus were first revealed by scanning electron microscopy and atomic force microscopy. Further analyses revealed that the fungal nanoparticles had an average size of from about 360 nm to about 370 nm, with a zeta potential of -33 mV at pH 6.0. They contained about 28 μg of glycosaminoglycan and about 550 μg of protein per mg of nanoparticles.

Interestingly, the nanoparticles from A. oligospora demonstrated potential capability as an immuno-stimulatory and antitumor agent, as disclosed herein. They significantly induced TNF-a secretion in RAW264.7 mouse macrophages, showed slight cytotoxicity to mouse melanoma B16BL6 and human lung cancer A549 cells, and demonstrated a synergistic cytotoxic effect upon conjugation with DOX against both cells. Therefore, they can play multifunctional roles in tumor immune-chemotherapy. In this respect, the synergistic antitumor efficacy for chemotherapy, drug delivery and immune regulation are expected in vivo.

[0074] The disclosed nanoparticles and FNPs can be used for cancer therapy as a multifunctional nanocarrier. A new isolation procedure was established to purify the FNPs through combining size exclusion chromatography (SEC) and weak anion exchanger (WAX), through which two highly purified FNP fractions, FNP1 and FNP2, were obtained. AFM imaging and DLS analysis showed that both the purified FNPs had a reduced diameter of from about 100 to about 200 nm compared to the crude FNPs with diameter of from about 100 to about 500 nm. SDS-PAGE and chemical assays showed that the polysaccharides including glycosaminoglycan are the main components in the purified FNPs. The nanoparticles were demonstrated to enhance the secretion of multiple proinflammatory cytokines and chemokines from macrophages and splenocytes, measured by ELISArray, suggesting the efficacy of the FNPs as a potential immunomodulator of biological responses in adjuvant antitumor therapy. MTT assay showed that both the purified FNPs had mild to moderate cytotoxicity against multiple tumor cells, but the FNP2 had stronger cytotoxic activity than the FNP1. The apoptotic assay and cell cycle analysis further demonstrated that the FNP2, not the FNP1, could inhibit cell proliferation via inducing apoptosis and arresting tumor cells at sub G0/G1 phase (FIG. 15). To test the combined cancer therapeutic effects, both FNPs formed pH-responsive nanocomplexes with chemo- drug, DOX, via the electrostatic interactions. Upon binding of DOX to the FNPs, it was found that the DOX-FNP2 complexes had higher cytotoxic activity than free DOX against multiple tumor cells, while the cytotoxic activity of the DOX-FNP1 complexes was weaker than free DOX. Interestingly, in a co-culture experiment where splenocytes were co-cultured with tumor cells, both nanocomplexes

demonstrated higher antitumor activity than free DOX, suggesting a synergistic effect between the immunostimulation of the FNPs and cytotoxicity of the nanocomplexes in vitro.

Drop Culture Method

[0075] Few studies have focused on the discovery of naturally occurring organic nanoparticles from the microbial world. Unlike higher organisms, microorganisms themselves are on the micro-scale. The colonial growth, development and appearance of microorganisms are largely susceptible to the environmental parameters, such as culture method, nutrients and temperature. As a result, the possible interferences from the medium components/ingredients, especially the particle-based components present in solid medium, add a degree of complexity for more sophisticated designs to discover the nanoparticles formed inside or secreted from living microbial cells.

Therefore, a proper culture system for discovering nanoparticles from microbial cultures without possible interfering from the culture medium is crucial to allow this type of study. The fungal sitting drop culture method was first established as shown in FIG. 1. A drop of medium was added onto a sterile cover slip and placed into a small Petri dish (3 cm). Humidity was maintained by filling a second small Petri dish with water, and placing both into a large Petri dish (10 cm). 50-100 conidia of A. oligospora from a stock culture on CMA dish were then inoculated into the drop and incubated at 25°C. The low nutrient medium supplemented with 1 mg/mL phe-va was used as the media for fungal growth. Germination took place during the first 24 hours post inoculation and after 3-4 days the first adhesive loop with diameter of about 20 to about 30 mm on the parent hyphae was observed. After this initial event, adhesive trap networks, consisting of one to several loops attached to each other in a 3D conformation, began to develop on the thin mycelium. The filamentous mycelium gradually grew to form a thick layer and the 3D traps increased both in size and number over a 5-15 day period. FIG. 2 shows images of the filamentous mycelia of A. oligospora and 3D-traps in both a hydrated and dehydrated state at day 5 post- inoculation. The drop culture system was incubated at 25°C for 15 days until the secreted nanoparticles were harvested.

[0076] A simple media, including five inorganic salts, two vitamins and one dipeptide was used, which prevented any particle contamination from the agar components present in solid media. Because the fungus was cultured in a drop of media on a cover slip, the growth of the fungus can be monitored using optical microscopy in situ and even the secreted biomaterials using SEM or AFM.

[0077] Additionally, the fungus used is aerobic and generally needs aeration or bubbling when cultured in liquid medium; however, the drop volume used in this system was 100-500 μΐ,, which allowed for significant gas exchange. Therefore this culture system is capable of growing fungal mycelia without any aeration or bubbling, and is adaptable for any aerobic fungal species. More importantly, this drop culture system made collection and preparation of the secreted nanomaterials easier, through washing or sonicating the mycelia.

[0078] Using this culture system and the liquid low nutrient medium, abundant hyphae and 3D traps formed, and some secretory nanoparticles were observed. It was confirmed that the nanoparticles secreted from A. oligospora were not related to the formation of the 3D traps, but instead were secreted from growing mycelia. Currently, little is known about how the nanoparticles are secreted from this fungus and what biological functions they play during the growth and development of the fungus. However, this study has confirmed that glycosaminoglycan (GAG) and protein were components of the nanoparticle samples. As well-known, most glycosaminoglycans are covalently attached to core proteins to form proteoglycans, and some newly formed basement membrane proteoglycans or secretory proteoglycans are transported outside via secretory granules from the Golgi apparatus in mammalian cells.

[0079] Even though the nanoparticles themselves have only a minimal cytotoxic activity, they are still useful for tumor therapy, especially when conjugated with certain antitumor drugs. In this respect, it is demonstrated herein that the disclosed nanoparticles or FNPs can function as a drug carrier by conjugating the antitumor drug DOX to the nanoparticles. It is known that carboxylic acid and sulphate groups are deprotonated at physiological pH, giving GAGs very high negative charge densities. Due to the existence of GAGs in the nanoparticles, it was assumed that there were carboxyl and sulphate groups on the nanoparticles. In order to demonstrate the nanoparticles as drug carriers for tumor therapy, doxorubicin was conjugated to these carboxyl groups via amide linkages (FIG. 19). Amide linkers are more stable than hydrazone or carbamate linkages in the case of conjugating DOX to different drug carriers in vitro and in vivo.

Immunostimulatory Activity of the Nanoparticles

[0080] GAGs are unbranched polysaccharides composed of repeating units of alternating uronic acids and amino sugars. Significant experimental evidence suggests that some polysaccharides from higher plants, mushrooms, lichens and algae possess an immunostimulatory activity both in vivo and in vitro. Some GAGs, such as heparin, heparan sulfate, chondroitin sulfate and their mimetics, have also been reported as potential cancer therapeutics, inhibiting tumor cell adhesion, migration, growth, and invasion in vitro. Other proteoglycans formed by covalently attaching GAGs to core proteins, such as decorin, seem to be effective potential therapeutics that reduced primary tumor growth by 70% and eliminated metastases in an orthotopic mammary carcinoma model as reported in Reed et al. , Oncogene

2005;24(6): 1104-10.

[0081 ] The immunostimulatory effect and antitumor activity of the nanoparticles disclosed herein were tested in vitro. One approach to evaluate potential

immunomodulating activity is the ability of a particular substance to induce cytokine production and inflammation mediators from immune cells. Macrophages and lymphocytes are representative immune cells that regulate innate and adaptive immunity of the host defense. Macrophages are the first cells to recognize invading foreign bodies and are central to cell-mediated and humoral immunity. The elevated secretion of cytokines, such as TNF-a, IL-6 and IL-12, from activated immune cells trigger other immune cells through various receptors including complement receptor (CR3), scavenger receptors, as well as TLR4 or TLR2/6 40. It was confirmed that the fungal nanoparticles induced a 3 to 39-fold increase in TNF-a secretion from

RAW264.7 macrophages in a dose-dependent manner. However, no significant induction in IL-12 secretion was observed from the activated macrophages. This indicates that the nanoparticles disclosed herein possess immunostimulatory effects. At the same time, examination of the cytotoxicity of the nanoparticles against RAW 264.7 macrophages by the MTT assay indicated that there was no cytotoxicity on RAW 264.7 macrophages. On the contrary, besides the macrophage-activating effects mentioned above, the nanoparticles unexpectedly enhanced the proliferation of RAW 264.7 macrophages in a dose-dependent manner at all concentrations examined during the 24-hour incubation. It was previously reported that TNF-a has no proliferation effects alone but decreases significantly the population doubling time for mouse macrophages stimulated by macrophage colony stimulating factor. The increased proliferation of the macrophages disclosed herein is believed to aid the modulation of macrophage immune function in vivo.

[0082] TNF-a is a Thl -biased cytokine and has been regarded as a potential anticancer agent for many years. It plays a key role in apoptosis, cell survival, inflammation and immunity, and has been shown critical for antitumor T cell immunity in mice. It can act synergistically with other drugs at the molecular level to trigger the apoptosis and dissociation of tumor vascular endothelial cells in cancer treatment. As a Th2 -biased cytokine, IL-6 plays key roles in T-cell-mediated immune responses, acting as a cofactor for T-cell proliferation, and as a growth inhibiting factor, the antitumor effect of IL-6 on multiple murine tumor in vivo has been reported (Kang et al, JBiomed Sci 1999;6: 142-4; Mule et al, J Immunol

1992;148:2622-9; Ishiguro et al, Cancer Immunol Immunother 2005;54: 1191-9). G- CSF can enhance the differentiation of stem cells in bone marrow, facilitate the mobilization of hematopoietic precursor cells into the bloodstream (Thomas et al., Curr Opin Hematol 2002;9: 183-9), and accelerate recovery from chemotherapy- induced myelosuppression (Voloshin et al, Blood 2011;118:3426-35). The synergistic antitumor effect of TNF-a and G-CSF has been established and antitumor effect of TNF was enhanced by combining with G-CSF in multiple tumors in vivo (Maeda et al, Jpn J Cancer Res 1993;84:921-7). As such, the elevated levels of TNF- , IL-6 and G-CSF secreted from the macrophages under the treatment with the nanoparticles disclosed herein are believed to be beneficial for direct or adjuvant anticancer therapy using the FNPs.

[0083] It has been reported that IL-1 has multiple effects, involving

immunomodulation, inflammation, wound healing, hematopoiesis, metabolism and the endocrine system (Veltri et al, Oncologist 1996;1 : 190-200). IL-1, including IL-la and β, also has a number of properties potentially useful in the treatment of cancer, including direct antiproliferative activity against certain human tumor cell lines and several murine tumors, the activation of effector cells in vitro, and the inhibition of tumor angiogenesis. In addition, IL-1 has the capacity to protect and restore the bone marrow from radiation or chemotherapy-induced injury. The cytokine IL-2 is known to be a T-cell growth factor, inducing clonal expansion of T cells following antigen stimulation, and is also important for the differentiation of CD4+ T cells into Thl and Th2 effector subsets. IL-2 has been used for the treatment of melanoma and renal cell carcinoma (Carmenate et al, J Immunol 2013;190:6230-8.), and recombinant human IL-2 is a potent cytokine and a FDA-approved anticancer drug (Chou et al, Cancer Immunol Immun 2013;62:597-603.). As such, the three cytokines, IL-la, IL-Ιβ and IL-2, with elevated levels in the supernatant of splenocytes after the treatment with the FNPs also favor the antitumor immunity in the cancer treatment. Additionally, in the treated splenocytes, only the crude fungal nanoparticle, FNP0, induced

significantly higher amount of IL-10, IL-17A, IFN-γ, and G-CSF as compared to the untreated control, while the purified FNPs did not show this activity. IFN-γ is a functionally pleiotropic cytokine, and has direct anti-proliferative effects on some tumor cell lines. Hence, like G-CSF, IFN-γ can also benefit for antitumor immunity. The bias in immunostimulatory activity between the purified FNPs and the crude FNPs are helpful to establish the antitumor immunity in vivo, because the elevated level of IL-10, an anti-inflammatory, immunosuppressive cytokine that favors tumor escape from immune surveillance (Jarnicki et al, J Immunol 2006;177:896-904), was observed in the supernatant of splenocytes treated with the crude fungal nanoparticles, FNPO. In addition, a higher amount of cytokine, IL-17A, was also observed from the treated splenocytes by the crude FNPO, and a low but significant increase in this cytokine was observed in the treated macrophage by the FNP2. Overall, these results suggest that the FNPs, especially the purified FNP1 and FNP2, can potentially modulate the immune cells to an activated state to induce an efficient antitumor response in the cancer immunotherapy.

[0084] Chemokines are small chemotactic cytokines, which can induce migration of leukocytes, activate inflammatory responses, and are implicated in the regulation of tumor development and growth (Luo et al., Cell Mol Immunol 2004;1 : 199-204). They can modulate tumor growth via regulating tumor-associated angiogenesis, activating host immunological responses, or direct inhibiting tumor cell proliferation, or modulating the neutrophils influx into the tumor tissue after treatment with chemo- drug during the immunochemotheray (Wang et al., Neoplasia 2009; 11 :793-803).

[0085] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

EXAMPLES

[0086] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0087] Efforts have been made to ensure accuracy with respect to numbers {e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions. [0088] Arthrobotrys oligospora (ATCC 24927), A549 human non-small-cell lung cancer cells (CCL-185), and RAW 264.7 murine macrophages (TIB-71) were obtained from the American Type Culture Collection (Manassas, VA). B16BL6 murine melanoma cells, MCF-7 human breast tumor cell line, and multidrug resistant cell line MCF-7/ADR were obtained from the National Cancer Institute-Central Repository (Frederick, MD). Splenocytes, derived from C57BL/6 mice, were purchased from the Allcells Company (Emeryville, CA). Corn Meal Agar (CM A), phe-val, thiamin-HCl, biotin, PBS, HEPES, 1, 9-dimethyl-methylene blue (DMMB), chondroitin sulfate (CS), Sephadex G75, DEAE-cellulose and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (St. Louis, MO). Doxorubicin hydrochloride (DOX) was purchased from Abeam (Combridge, MA). LysoTracker Green DND-26 and Hoechst 33342 were purchased from Invitrogen Life

Technologies (Grand Island, NY). Fetal bovine serum, DMEM medium and RPMI 1640 medium were purchased from Mediatech (Manassas, VA). Penicillin (10000 units/ml)-streptomycin (10000 μg/ml) solution was obtained from MP biomedicals (Solon, OH).

[0089] Scanning Electron Microscopy (SEM) were performed on LEO 1525 (High resolution FE-SEM system, Germany). Particle size, size distribution and zeta potential of the dialyzed nanoparticles were determined at 25°C using a Malvern Zetasizer, NANO ZS (Malvern Instruments Limited, UK), with a He-Ne laser (wavelength of 633 nm) and a detector angle of 173°. The sample NONP-DOX conjugates were measured using a dynamic light scattering (DLS) method. All samples were measured in triplicate. The amount of sulfated glycosaminoglycans in the nanoparticle samples were quantified using a Proteoglycan Detection Kit (1,9- dimethylmethylene blue, Astarte Biologies, Redmond, WA) according to the manufacturer's procedure. Similarly, the concentration of protein in the samples was quantitatively determined using the BCA Kit (Pierce, Rockford, IL) according to the manufacturer's protocol. The protein in the nanobiomaterials was also qualitatively measured with SDS-PAGE.

[0090] All the values were presented as mean ± standard deviation (S.D.) of at least three independent measurements. Statistical significance was tested by one-way ANOVA followed by a Student's t test for multiple comparison tests. A p value of <0.05 was considered statistically significant.

Example 1: Arthrobotrys oligospora Culture and Nanoparticle Formation

[0091] Freeze-dried A. oligospora was first inoculated into CMA medium (pH 7.0) and incubated at 25°C for 3-15 days, resulting in mycelia development and sufficient conidia production. After 10-15 days, a piece of the fungus was cut, transferred to a silicon substrate, and dried for SEM observation. The conidia were collected from the above stock cultures using 0.02% Tween-80, and aliquots of the conidia suspension were stored at -20°C. A sitting drop culture method was used. Specifically, a drop of medium was added onto a sterile cover slip and placed into a small Petri dish (3 cm), and then 10 of conidia suspension (about 50-100 conidia) were inoculated into the media droplet and incubated at 25°C for 3-15 days. Humidity was maintained by filling a second small Petri dish with water, and placing it alongside the first Petri dish into a large Petri dish (10 cm). A modified low nutrient medium (LNM) was used for the fungal drop culture. The ingredients of the low nutrient medium are listed in Table 1.

Table 1

Ingredients Concentration

MgSCv7H 2 0 0.2 mg/mL

KC1 1 mg/mL

FeCl 3 -6H 2 0 0.03 mg/mL

ZnSCv7H 2 0 0.88 μg/mL

MnS0 4 H 2 0 0.3 μg/mL

thiamin-HCl 2 μg/mL

biotin 0.05 μg/mL

[0092] In order to test the possible source of the nanoparticles, different media were used in the drop culture system, and the amounts of nanoparticles produced were compared. For instance, the LNM supplemented with 1 mg/mL phe-val was used to grow the predatory stage of the fungus (formation of 3D adhesive capture traps), or supplemented with 12 mM phosphate buffer for growth of the saprophytic stage of the fungus (only mycelia growth without trap formation). In experiments testing the effect of LNM ingredients on the nanoparticle secretion, distilled water (pH 7.0) was also used to completely or partially replace the LNM. The effect of 3D-traps, mycelia and medium ingredients on the nanoparticle secretion from the fungus is listed in Table 2 below.

Table 2

3D- Mycelia a Nanoparticles traps a (GAG, μ ξ γ

LNM + dipeptide +++ +++ +++ (8.39±0.15)

LNM + PO 4 - +++ +++ (8.44±0.27)

LNM + +++ +++ (8.21±0.11)

LNM + dipeptide (initial media) + ++ ++ (6.17±0.34) /water (subsequent replenishment)

Water - + + (2.14±0.05)

Note: a the amount of 3D-traps and mycelia observed by optical microscopy;

b the GAG amounts stand for the amount of the nanoparticle.

[0093] As shown in Table 2, when the LNM was supplemented with 1 mg/mL phe-val, the predatory stage of the fungus developed, resulting in abundant 3D adhesive traps as shown in FIG. 2. When the LNM was supplemented with 12 mM phosphate buffer, only the saprophytic stage of the fungus developed, resulting in comparable mycelia growth without trap formation. However, the nanoparticles produced from the above two media did not show significant differences in GAG concentration. In the case of the LNM without phe-val, the fungus developed sparse 3D traps, but abundant hyphae growth. It also produced the comparable amount of the nanoparticles compared to the LNM with phe-val or phosphate buffer. However, when LNM/phe-val was initially used as the medium, and then changed to pure water, the 3D traps and hyphae developed in the drop culture system were less sparse than using LNM/phe-val medium. Accordingly, the fungus produced fewer amounts of nanoparticles. If only water was used as a medium, the fungus grew slowly, producing the fewest hyphae and no traps. Consequently these samples had the fewest amount of nanoparticles. Based on this data, it can be concluded that the nanoparticles were not related to 3D-trap formation, but instead to the growth of mycelia. [0094] The fungal hyphae development and the formation of 3D traps were monitored in situ using inverted optical microscopy and the results are plotted in FIG. 2A and FIG. 2B with arrows indicating the 3D-traps and scale bar representing 200 μιη. A. oligospora growth in a medium drop on the cover slip on day 5 post- inoculation is shown in FIG. 2A. The air-dried A. oligospora mycelia with 3D-traps on the cover slip on day 5 post-inoculation is shown in FIG. 2B. As shown in FIGs. 2A & 2B, abundant nanoparticles were observed on the surfaces of the hyphae and 3D-traps. Additionally, some nanoparticles were observed on the surface of the silicon substrate.

[0095] The drop culture system was incubated at 25°C for 15 days until the secreted nanoparticles were harvested. Specifically, after a 15-day incubation, the media was removed, the mycelia on the cover slip were first washed 10 times using 1 mL of distilled water, and the wash water was collected and designated as sample NONP-W. The washed mycelia on the cover slip were also collected in lmL distilled water and then were sonicated in a water-bath sonicator (model 750D, VWR) at RT for 30 min. The sonicated sample was spun down at 1000 rpm for 5 min and the supernatant was collected and designated as sample NONP-S. Both sample NONP-W and NONP-S were filtered through a 1 μιη syringe filter (Nylon membrane,

Whatman, Florham Park, NJ) and then dialyzed using CE dialysis tubing or cellulose acetate dialysis membrane (MWCO 300K, Spectrum Labs, CA) against distilled water for 3 days to remove small organic molecules. 10 of both dialyzed NONP-W and NONP-S were then transferred onto silicon wafers, dried and directly examined using SEM. The nanoparticles were also visualized using an Agilent 6000 ILM/AFM atomic force microscope (Agilent Technologies, Santa Clara, CA) by placing and air- drying ΙΟμί of the samples on glass cover slips. The operation was performed in AC mode, using an ACTA Probe from AppNano (Santa Clara, CA) controlled with the PICOVIEW™ software package. The SEM/AFM images of nanoparticles generated from A. oligospora cultured in the drop culture system were plotted in FIG. 3. FIG. 3 A shows nanoparticles observed in situ on the surface of hyphae and FIG. 3B shows the 3-D traps of A. oligospora using SEM on the silicon wafer. FIG. 3C shows SEM images of nanoparticles NONP-W prepared by washing the fungal mycelia and FIG. 3D is an enlarged view of FIG. 3C. FIG. 3E shows the AFM images of nanoparticles NONP-S prepared by washing the fungal mycelia and FIG. 3F is an enlarged view of FIG. 3E. As shown in FIGs. 3C and 3D, the nanoparticles prepared by washing, NONP-W, had a diameter of 200-300 nm and were spherical in shape when analyzed by SEM. AFM revealed a similar finding, with nanoparticles ranging from 300-500 nm in diameter (FIGs. 3E-F). The nanoparticles prepared through sonication, NONP- S, were not significantly different from the washed nanoparticles.

Example 2: Conjugation of Nanoparticles with Doxorubicin (DOX)

[0096] In order to test the potential of the nanoparticles to be used as drug carriers for tumor therapy, an antitumor drug, doxorubicin (DOX), was conjugated to the NONP-W samples. Conjugation of DOX to the nanoparticles through amide bonds between the 3 '-amine of DOX and the free carboxyl groups of the nanoparticles was achieved using EDC and sulfo-NHS as the coupling agents. Briefly, the nanoparticles (0.1 mg) were suspended in 0.5 mL water and mixed with EDC (0.4 mg) and sulfo- NHS (0.1 mg). The solution was incubated at ambient temperature for 30 min to modify the carboxyl groups of the nanoparticles with sulfo-NHS. After that, DOX (0.26 mg) was added to the activated solution, and stirred overnight to complete the reaction. Finally, the resulting solution was poured into the CE dialysis tube (MWCO 300K) and dialyzed against deionized water for 3 days. After lyophilization, the obtained NONP-DOX conjugate was characterized by FTIR analysis (Varian). Three replicates were carried out to assess the average DOX content in NONP-DOX conjugate and the bioconjugation efficiency (%). The particle size and zeta potential of NONP-DOX conjugates were also measured. The bioconjugation efficiency for the NONP-DOX conjugates was 29% under current reaction conditions.

Example 3: Characterization of NONP-W, NONP-S, and NONP-DOX

[0097] The FTIR spectra of NONP-W before and after conjugation to DOX were plotted in FIG. 4. Specifically, Fourier transform infrared (FTIR) spectra of (1) NONP-W, (2) NONP-DOX conjugates and (3) free DOX were measured. The band around 1730 cm "1 was assigned to the stretching vibration of the carbonyl group at the 13-keto position of DOX, the band around 1617 and 1581 cm "1 are assigned to the stretching vibration of two carbonyl groups of the anthracene ring, respectively, and the band around 1114 and 1073 cm "1 was assigned to the stretching vibration of C-0 bonds, and a band at 1284 cm "1 due to the skeleton vibration of the DOX molecular. In the case of NONP-W, there are the presence of the amide bond signal (1634 and 1555cm "1 ) within the nanoparticle itself due to the existence of glycosaminoglycan and protein in the nanoparticles. After conjugation, the new peak of amide bond signal was not apparently observed due to the presence of amide bond signal (1634 and 1554 cm "1 ) within nanoparticle itself. However, the band near 1752, 1265 and 1101 cm "1 for the uncongjugated NONP-W shifted to 1736, 1236 and 1044 cm "1 upon conjugation.

[0098] The particle size and zeta potential of the sample NONP-W, sample

NONP-S, and the sample NONP-DOX conjugates were measured using a DLS method. The average particle sizes of both nanoparticles were about 360-370 nm. The average nanoparticle size of NONP-DOX was about 380 nm. There were therefore no significant differences in size for the NONP-DOX conjugates compared to both nanoparticles. However, the zeta potential for the NONP-DOX conjugates increased to -20 mv from -33 mV at pH 6.0 of unconjugated nanoparticles.

[0099] To determine the presence of glycosaminoglycan and protein in the nanoparticles, the dialyzed NONP-W and NONP-S samples were lyophilized and weighed and measured using commercially available proteoglycan and BCA kits respectively. The results are summarized in Table 3 below. As shown in Table 3, there were about 30 μg of GAG and about 550 μg of protein in 1 mg of lyophilized nanoparticles. In addition, SDS-PAGE analysis confirmed that there were two main proteins with MWs of 110 KDa and 80 KDa in both nanoparticle samples.

Table 3

Particle Size Zeta Potential GAG Protein (nm) (mV) g/m ) g/mg)

NONP-W 371.2±74.59 -33.2±5.55 28.03±1.32 544.73±16.70

NONP-S 369.0±67.49 -33.9±5.77 28.22±5.39 554.42±22.93

NONP-DOX 380.9±60.13 -20.8±5.37 — —

Example 4: Immunostimulatory Activity of NONP-W and NONP-S

[00100] Mouse macrophage cell line RAW264.7 was used to measure the potential immunostimulatory effect of the nanoparticles. The immunostimulatory potential of the NONP-W and NONP-S samples was tested on RAW 264.7 macrophages using dose-titration (0.5-25 μg/ml) assays. Briefly, the RAW 264.7 cells were grown in DMEM medium supplemented with 10% heat-inactivated FBS and 1% penicillin- streptomycin at 37 °C. The cells were then seeded onto 12-well culture plates at a density of 5x 10 6 cells/mL and cultured for 24 hours. The cells were then washed three times with 0.5 mL PBS, before addition of NONP-W and NONP-S samples at various concentrations. After addition of the test samples, the cells were incubated for 24 hours, and the supernatants were collected and stored at -20°C. The levels of TNF-a and IL-12 in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA) using an OptEIA ELISA Set (BD, San Jose, CA). The proliferation rate was measured using the MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay after removing the cytokine-containing supernatant at 24h post incubation.

[00101] Results are expressed as mean ±SD. *P < 0.05,†P < 0.01 , significantly different from the control and plotted in FIG. 5. Specifically, FIG. 5 A shows the TNF-a concentration in the culture media of RAW264.7 cells treated with the nanoparticles for 24 hours. In both cases, NONP-W and NONP-S, the RAW264.7 cells secreted significantly greater amounts of TNF-a (about 3 -to 39-fold) than the control treatment, in a dose-dependent manner. There were no significant differences in TNF-a induction between the NONP-W and NONP-S samples. In addition, there was no increase in IL-12 secretion from any of the samples tested as shown in FIG. 5B. Surprisingly the proliferation of the RAW 264.7 macrophages increased in a dose dependent manner with the addition of the NONP samples compared to the control sample as shown in FIG. 5C.

Example 5: MTT Assay of Nanoparticles for Cytotoxicity

[00102] Using the A549 human non-small-cell lung cancer and B 16BL6 mouse melanoma cell lines, the cytotoxicity of the fungal nanoparticles was further evaluated. The MTT assay was utilized to assess cytotoxicity of the nanoparticles. Both tumor cells were treated with NONP-W and NONP-S nanoparticles at the concentration range of 2-100 μg/mL or with the NONP-DOX conjugates at the DOX concentration range of 0.1-10000 nM for 48 hr. Briefly, lxlO 4 tumor cells (murine melanoma B 16BL6 and human non-small-cell lung cancer A549) were seeded in 96- well plates in 100 DMEM. Serial dilutions of nanoparticles were added to the plate and incubated at 37°C in 5% C0 2 for 48 hr. 10 μΐ ^ of MTT stock solution (5 mg/mL in PBS, pH 7.4) were then added into the wells and the plates were incubated at 37°C for another 4 hours. The medium was then removed and ΙΟΟμΙ of DMSO was added to each well to solubilize the dye. The absorbance was measured using a microplate reader (Bio-Tek μ Quant) at 570 nm. The inhibitory effects of both samples, NONP- W and NONP-S, on both cells in dose-dependent manners were observed at the NONP concentration examined. The results were plotted in FIGs. 6A-B. As shown in FIGs. 6 A-B, both nanoparticle samples showed dose-dependent cytotoxicity on both tumor cell lines. At a concentration of 100 μg/mL, the nanoparticles inhibited cell proliferation by 35-45 %. At a concentration of 2 μg/mL, the inhibition fell to 5-10%, and no significant differences were seen for both cell lines at that concentration.

[00103] The sigmoidal dose-response curves were fitted and the concentration of NONP-DOX conjugates and free DOX that inhibited cell survival by 50% (IC 50 ) was determined from cell survival plots using the "DoseResp" function of OriginPro 8.0 and the results plotted in FIG. 6 C-D. As shown in FIGs. 6 C-D, the NONP-DOX conjugates showed higher inhibitory effects on both cells in dose-dependent manners, compared to free DOX. The IC 50 values for NONP-DOX conjugates and free DOX are listed in Table 4. Compared to the free DOX, the IC 50 values of the NONP-DOX conjugates were 1.6-fold lower in A549 cells and 1.8-fold lower in B16BL6 cells. The IC 50 of the nanoparticles for both tumor cell lines was estimated to be greater than 100μg/mL, indicating that the nanoparticles had only a slight antitumor activity compared to some cytotoxic antitumor drugs. The studies demonstrated a synergistic cytotoxic effect when DOX was conjugated to the NONP. At an IC 50 of free DOX for both cells (about 254 nM for B16BL6, and about 552 nM for A549, Table 4), the free DOX inhibited cell proliferation by 50 %, whereas the NONP-DOX conjugates showed 61 % and 68% inhibition on the A549 and B16B16 cells, respectively.

According to the bioconjugation efficacy (29%), the concentration of NONP-W in the NONP-DOX conjugates at the respective IC 50 of free DOX for the A549 and B16B16 cells was calculated to be around 0.51 and 1.1 μg/mL; however, almost no significant inhibition effect on both cells was observed at that concentration for nanoparticles themselves (as shown in FIG. 6A-B). This indicates that NONP and DOX in the conjugated nanoparticles exerted synergistic cytotoxic effects, which led to the IC 50 values 1.6-1.8 folds lower in A549 cells and B16BL6 cells. Table 4

Lung cancer Melanoma

A549 cells B16BL6 cells

Free DOX 551.71±36.72 254.22±11.35

NONP-DOX conjugates 342.34±57.17 139.93±28.30

NONP control >100 μg/mL >100 μg/mL

Note: *P<0.05 and†P<0.01, as compared with the free DOX.

Example 6: NONP Cellular Uptake and Confocal Microscopic Study

[00104] The cellular uptake of DOX was quantified using Flow cytometry analysis. Briefly, human lung cancer A549 cells and mouse melanoma B16BL6 cells were seeded into 6-well plates at densities of 1 x 10 6 cells/mL, and incubated at 37°C until 70% confluence was reached. Free DOX solution or NONP-DOX conjugates at DOX concentration of 10 μΜ were then added and incubated for 4 hours at 37°C. The medium was aspirated and cells were rinsed with cold PBS three times. Flow cytometry analysis was immediately carried out on an Epics XL Analyzer (Beckman Coulter Inc., Brea, CA) by collecting 20000 events for each sample and the cell associated fluorescence was measured. Each experiment was performed in triplicate. As shown in FIG. 7, after a 4-hour incubation, there was no significant difference in the DOX fluorescence for free DOX and NONP-DOX conjugates at a DOX concentration of 10 μΜ. FIG. 7A shows the flow cytometry analysis for cellular uptake of NONP-DOX conjugates in human lung cancer A549 cells and FIG. 7B shows the uptake in mouse melanoma B16BL6 cells. Statistical analysis of the mean DOX fluorescence associated with the cells was compared, and no significant differences between DOX and NONP-DOX conjugates were observed for respective cell line as shown in FIG. 7C.

[00105] Confocal microscopy was used to investigate the intracellular distribution in the tumor cells treated with NONP-DOX conjugates and free DOX. The cells were grown on cover slips to 50%> confluence and incubated with samples at DOX concentrations of 10 μΜ at 37°C for 4 hours. To observe the intracellular distribution of the DOX-loaded NONP, the cells were incubated with acidic endolysosomes- selective dye LysoTracker green (100 nM) and nucleus-selective dye Hoechst 33342 blue (4 μΜ) for 30 min prior to visualization by confocal microscopy for endosomes/lysosomes (endolysosome) and nuclear labeling, respectively. The cells were then washed three times with PBS, and immediately imaged by a FluoView FV1000 Confocal Microscope (Olympus, Japan). As shown in FIG. 8, different intracellular distribution of NONP-DOX conjugates and free DOX was observed in both cells after a 4-hour treatment. FIG. 8A shows the images of A549 cells and FIG. 8B shows the images of B16BL6 cells. Scale bar represents 10 μιη. The majority of DOX in both cells treated with NONP-DOX conjugates were predominantly located in the endosomal/lysosomal compartment, whereas both cells treated with free DOX showed that the major fraction of DOX was localized to the cytoplasm, outside of the endolysosomes, suggesting that the nanoparticles were taken up by endocytosis. In addition, a small fraction of DOX was seen in the nucleus of B16BL6 cells treated with free DOX, whereas the treatment of NONP-DOX conjugates did not result in obvious nuclear distribution (FIG. 8B). In the case of A549 cells, no significant DOX fluorescence was seen in the nucleus for both treatments (FIG. 8A). As indicated in FIG. 7, there was no difference in the intracellular DOX fluorescence from the free DOX and NONP-DOX conjugates, indicating that conjugation of DOX to

nanoparticles did not decrease DOX uptake by both tumor cell lines even though there was a different sub-cellular distribution.

Example 7: Arthrobotrys oligospora Culture and FNPs Fractionation

[00106] The fungal sitting drop culture method disclosed in Example 1 with some modifications to scale-up production and improve purification quality was used to produce the nanoparticles from A. oligospora. Briefly, conidia suspension (about 1000-2000 conidia in 200 μί) was inoculated into the media droplet (about 500μ1) and incubated at 25°C for 7 days. The isolation procedure was then performed.

Specifically, first, the mycelia developed on the cover slip were washed over 10 times using distilled water. The collected water containing nanoparticles were then filtered through a 0.2 μιη syringe filter (cellulose acetate, VWR, Radnor, PA). The FNPs were then desalted by using a size exclusion chromatography (SEC, Sephadex G75, 15 mmx70 mm). The desalted FNPs were designated as FNP0, which is a crude sample. These crude FNPs have been determined to be a spheroidal shape with diameter of 100-500 nm. To further purify the FNP0, a weak anion-exchange chromatography (WAX, DEAE-cellulose, 10 mmx70 mm) of FNP0 was performed. The DEAE- cellulose columns were eluted in a stepwise fashion with 0.1 M, 0.2 M, 0.3 M, 0.5 M and 1.0 M NaCl. As indicated in previous examples, glycosaminoglycan (GAG) has been determined to be one of the main components in the FNPs. Thus, the

colorimetric assay (λ 525 nm) for GAG with 1, 9-dimethyl-methylene blue was used to monitor the FNPs in the eluates from the SEC or DEAE-cellulose column. The elution profiles of the FNPs, reflected from GAG concentration versus elution volumes were obtained and plotted in FIG. 9. FIG. 9A is the elution profile of FNP0 and FIG. 9B is the elution profile of FNPl and FNP2. Based on the elution profile of FIG. 9B, two FNP fractions eluted from 0.5M NaCl and 1.0M NaCl were collected and designated as the FNPl and FNP2, respectively. The collected FNPl and FNP2 from WAX column were subjected to the Sephadex G-75 column for desalting. The desalted FNP0, FNPl and FNP2 were concentrated to final volume of 150 using a centrifugal filter tube (Amicon Ultra- 15 100K, Merck Millipore, Ireland).

[00107] To characterize nanomorphology and particle size of the FNPs, the samples were analyzed using AFM (MFP-3D, Asylum Research, Santa Barbara, CA). Briefly, 10 of the particle solution was air-dried on a glass cover slip, and analyzed in AC mode based on the software Igor Pro from Wavemetrics and an ACTA Probe from AppNano (Santa Clara, CA) at room temperature. The

nanoparticle samples were also analyzed by DLS and electrophoretic light scattering (ELS), using a Zetasizer Nano (Malvern Instruments Ltd, Worcestershine, UK), to determine the size distribution and zeta potential of the nanoparticles in solution. To qualitatively determine the chemical components in the nanoparticles, SDS-PAGE was used for staining the GAG and neutral polysaccharides in the nanoparticles using Alsian blue and PAS reagents (Thermo Scientific, MI), respectively. To quantitatively determine the chemical components in the nanoparticles, total amounts of

polysaccharides were measured using anthrone-sulfuric acid assay. The amount of GAG in each sample was determined by a Proteoglycan Detection Kit (1,9- dimethylmethylene blue, Astarte Biologies, Redmond, WA), and the uronic acid in the nanoparticles was determine using carbozole assay. Meanwhile, the concentration of proteins in the samples was quantitatively determined by the BCA protein assay (Pierce, Rockford, IL) following the manufacturer's instructions. The results from the physicochemical characterizations of the FNPs are listed in Table 5 below. Table 5

Size Zeta potential Protein GAG Uronic acid Total Sugar ( (mV) fcg/mL*) ^g/mL*) ^g/mL*) (μg/mL*)

FNPO 294.2 ± 152.3 -30.7 ± 9.1 661.1 ±10.7 187.6 ±10.7 162.6±23.1 410.1±6.4

FNP1 147.5 ± 78.4 -26.9 ± 6.9 86.8 ±6.3 296.5 ±38.1 153.9±10.8 506.2±25.2

FNP2 148.5 ± 67.4 -32.1 ± 7.6 3.7 ±0.7 98.7 ±7.4 40.4±7.7 162.7±8.5

Note: * Concentrations of protein, glycosaminoglycan (GAG), uronic acid and sugar in the FNPs were determined after the FNPs were concentrated to 150 μΐ ^ using a spin filter. Therefore, the unit here is designated as μg/mL nanoparticle solution instead of μg/mg freeze-dried nanoparticles. The FNPO was prepared using one batch of fungal culture (40 small disks), and the purified FNP1 and FNP2 were prepared using three batches of fungal culture (120 small disks).

[00108] AFM images (A-B, D-E and G-H) and size distributions (C, F and I) of the FNPO, FNP1 and FNP2 are plotted in FIG. 10. As shown in FIGs. 10A-B, spheroidal nanoparticles FNPO with a diameter of 100-1000 nm were observed. The

nanoparticles FNPO showed average size of about 300 nm in FIG. IOC and negative zeta potential of about 30 mV (Table 5) determined by DSL and ESL. As shown in FIG. 10D-F, FNP1 comprises spheroidal nanoparticles with about 150 nm in

hydrodynamic diameter and in FIG. 10G-I, FNP2, showed similar hydrodynamic size with spheroidal morphology. However, as shown in Table 5, the FNP1 had zeta potential of about -27 mV, which is lower than that of FNP2 (about -32m V). The difference in zeta potentials for both FNP fractions is consistent with their elution profiles through the WAX column. Due to relatively lower surface charge of the

FNP1 fraction, it was eluted more easily at low concentration of NaCl than FNP2 fraction. Therefore, at low salt concentration, i.e. 0.5M NaCl, the FNP1 was first eluted, and then the FNP2 was selectively eluted at higher concentration, i.e. 1.0M

NaCl.

[00109] Apart from the differences in morphology, sizes and zeta potentials, SDS- PAGE analysis of polysaccharides in the FNPs using Alcian blue staining and PAS staining were also carried out and the results are plotted in FIG. 11. As shown in FIG. 11 A, Alcian blue was used for glycosaminoglycan (GAG) staining and in FIG. 1 IB, PAS was used for neutral polysaccharide staining. As shown in FIG. 11 A-B,

glycosaminoglycan (GAG) and neutral polysaccharides were demonstrated to be the major components for both FNP1 and FNP2 fractions, which are similar to the chemical components of the crude FNPO.

[00110] The quantitative data for GAG, uronic acid and total sugars in the three FNPs are listed in Table 5. For the protein component in the three FNP samples, the ratio of protein to total sugar in the FNPO was much higher than those in the TNP1 and TNP2 (Table 5), indicating that most proteins in the crude FNPO were free and unbound. These proteins were not bound to the FNPs, leading to complete wash away through the purification process with lower salt concentration (< 0.5M NaCl).

Compared to the amount of total sugar, the purified FNP1 and FNP2 had much lower ratios of proteins to total sugars. It is likely that most GAGs are covalently attached to core proteins to form proteoglycan in the nanoparticles. Independent of the chemically bound or physically associated proteins, the polysaccharides, including acidic GAG and neutral polysaccharides, are the main chemical components in the purified FNPs. Example 8: Immunostimulatory Activity of FNPs

[00111] The mouse macrophage RAW 264.7 cells and splenocytes derived from C57BL/6 mice were cultured in DMEM and RPMI 1640 culture media, respectively. Both media were supplemented with 10% FBS and 1% penicillin- streptomycin at 37°C in 5% C0 2 . The cells were plated in 12-well plates at a density of 5x 10 6 cells/mL, treated with the FNPs at the GAG concentration of 5 μg/mL. After a 24- hour incubation, the supernatants were collected for ELISArray. Mouse common cytokines and chemokines multi-analyte ELISArray kits (SABiosciences Corporation, Frederick, MD) were used to determine 12 cytokines (IL-IA, IL-IB, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17A, IFNy, TNFa, G-CSF, and GM-CSF) and 12 chemokines (RANTES, MCP-1, MIP-la, MIP-lb, SDF-1, IP-10, MIG, Eotaxin, TARC, MDC, KC, and 6Ckine) in the supernatants following the manufacturer's instructions. The concentration of nitric oxide (NO) in the supernatants of both cells treated with the FNP samples were also determined using Griess assay, as described by Tincer et al. , Biomaterials 2011;32:4275-82. Effects of the FNPs on the secretion of cytokines (A and B), chemokines (C and D) and nitric oxide (E and F) from RAW 264.7 macrophage cells (A, C and E) and splenocytes (B, D and F) are plotted in FIG. 12. The results are expressed as mean ±SD. *P < 0.05,†P < 0.01, significantly different from the controls. [00112] The studies in Example 4 above have shown that the FNPs could induce the secretion of TNF-a from a macrophage cell line RAW264.7 in a dose-dependent manner, indicating the potential of antitumor immunity of the FNPs. In this example, the macrophage stimulatory activity of the purified FNPs (FNP1 and FNP2) was further studied using the cultured mouse macrophage RAW 264.7. As shown in FIG. 12 A, after a 24-hour incubation of the FNP -treated macrophage RAW264.7 at the GAG concentration of 5 μg/mL, ELISArray showed significantly elevated levels of IL-6, TNF-a, and G-CSF from the macrophage treated by the three FNP samples, FNP0, FNP1 and FNP2, as compared to the untreated cells. Of the panel of 12 cytokines assayed, except for the above three cytokines, low but statistically significant increases in IL-l and IL-17A were also detected for FNP1 and FNP2 in the supernatant of the treated macrophage, respectively.

[00113] The immunostimulation effects of the FNPs was further evaluated using primary splenocytes isolated from C57BL/6 mice, which have all types of immune cells and the cross talk between immunocytes, including macrophage and T cells. As shown in FIG. 12B, significantly higher amount of IL-6 and TNF-a were detected in the culture supernatant of the splenocytes treated with the three FNP samples in comparison with the untreated control. Other stimulatory cytokines, including IL-la, IL-Ιβ and IL-2, were also secreted in a low, but significantly higher amount from the treated groups with the three FNP samples.

[00114] Subsequently, the production of chemokines by the FNPs in both macrophages and splenocytes were investigated. As shown in FIG. 12C, of the panel of 12 chemokines assayed, RANTES, MCP-1 and IP- 10 were highly induced in the treated macrophages by the three FNPs, and low but statistically significant increases in MDC was also observed in the FNP2-treated macrophages. It has been reported that RANTES enhances anti-tumor immunity in a mouse model in part through direct T cell effector recruitment (Mule et ah, Hum Gene Ther 1996;7: 1545-53; Bacon et ah, Science 1995;269: 1727-30). MCP-1 has been reported to augment the antitumor effects by promoting lymphocyte infiltration into the tumor and subsequent cytokine production (Nakasone et al., Am J Pathol 2012;180:365-74). IP-10 has been demonstrated to elicit strong anti-tumor and anti-metastatic properties, and its immunological properties appear to be dependent on the attraction of monocytes and T lymphocytes (Keyser et al, Exp Dermatol 2004;13:380-90) while MDC is chemotactic for a variety of leukocytes, and has been shown to be involved in Th-2 mediated cellular immunity (Lee et al., J Immunother 2003;26: 117-29). For the activation of the splenocytes, except for RENTES, MCP-1, IP-10 and MDC, the elevated levels of the MIP-la, MIP-lb, TARC, and KC were also observed after treatment with the three FNPs as compared to untreated control as shown in FIG. 12D. It has been demonstrated that MIP-la showed potent anti-tumor effect after intravenous administration along with intra-tumor injection of certain adjuvants (Nakano et al, Int Immunopharmacol 2007;7:845-57), while MIP-lb is a chemokine which can chemoattract T cells and NK cells, inducing efficient antitumor responses in a pre-established tumor model (Luo et al. , Cell Mol Immunol 2004; 1 : 199-204). In conjunction with RANTES, TARC, which mediate the chemoattraction of both antitumor- specific effector T cells, has been demonstrated to enhance the antitumor immune effects of GM-CSF (Inoue et al, Cancer Immunol Immun 2008;57: 1399-411; Ghia et al, Blood 2001;98:533-40). The production of chemokine KC can amplify filtration of inflammatory cells, creating a more sustained antivascular action (Wang et al, Neoplasia 2009; 11 :793-803). Given the elevated cytokines and chemokines from both macrophage and splenocytes, the results here provide that the favorable antitumor immunity in vivo are established by stimulating different immunocytes, such as macrophages and splenocytes, with the fungal nanoparticles, especially the purified FNPs, FNP1 and FNP2.

[00115] Apart from multiple cytokines and chemokines, the bactericidal mediator, nitric oxide (NO), stimulated by the FNPs was further evaluated on both macrophages and splenocytes and results are plotted in FIGs. 12E-F. As shown in FIG. 12E-F, compared to the untreated cells, significantly elevated levels of NO were observed in both macrophages and splenocytes treated with the FNPs. NO is an important regulator and mediator of macrophage-directed cytotoxicity against tumor cells and microbes (Tincer et al, Biomaterials 2011;32:4275-82). Significantly higher amounts of NO production from the treated macrophages and splenocytes substantiate the anticancer immunity in the cancer immunotherapy using the polysaccharide-based nanoparticles secreted from fungi disclosed herein. Example 9: Formation and Characterization of DOX-FNP complexes

[00116] To use the purified FNPs as a nanocarrier for chemo-drug delivery by implementing the synergistic effect between the immunostimulation from FNPs and cytotoxicity from both the FNPs and chemo-drugs, DOX was chosen as a model chemo-drug to form a DOX-FNP nanocomplex. To prepare the complexes formed by the DOX and the FNPs, 60 μΐ. of DOX (3mM) was mixed with 100-200 μΐ. of FNP samples (containing about 15 μg GAG in each sample) in 20 mM HEPES buffer at pH 7.0, and then incubated at room temperature overnight. The precipitates formed by the DOX and the FNPs were then centrifuged at 10,000 rpm for 10 min. The precipitates were dispersed in 500 μΐ, PBS buffer, and then sonicated for 10 min in a bath sonicator (Aquasonic 7500, VWR). The amount of DOX in the dispersed precipitates was quantified by measuring UV absorbance at 480 nm and the entrapment ratios of DOX in the complexes were calculated as previously reported (Wang et al., Int JPharm 2012;422:409-17). To determine stability of DOX in the complexes in the PBS buffer, the dispersed complexes were applied to Sephadex G75 column. The first peak (standing for the stable complexes) and the second peak (standing for free DOX) were collected for quantification. The nanomorphology, particle size, and zeta potential of the DOX-FNP complexes were characterized using AFM, DLS, and ELS analysis.

[00117] DOX are efficiently bound to both FNP 1 and FNP2, when mixing DOX and the FNPs in 20 mM HEPES buffer at pH 7.0 in this example. Due to negative surface charges of the FNPl and FNP2 as shown in Table 5, DOX that carries positive charges from deprotonation of the amino group at pH7.0 in HEPES buffer (Wang et al, Pharm Res 2013; doi: 10.1166/jbn.2013.1724) bound to the FNPs via electrostatic interactions. The binding between DOX and the FNPs was highly efficient that leads to formation of precipitates overnight after centrifugation at 10,000 rpm for 10 min as shown in FIG. 13 A. The collected precipitates were then dispersed in PBS buffer (pH 7.0), and nano-sized DOX-FNP complexes were formed. The physical characteristics of the complexes were measured and the results are shown in Table 6 below.

Table 6

Size Zeta potential

Entrapment ratio (nm) (mV) DOX-FNP1 194.5±79.5 -22.2±7.48 77.4%±2.4%

DOX-FNP2 186.9±89.7 -24.24±5.95 72.2%±0.7%

Free DOX

[00118] As shown in Table 6, for both DOX-FNP1 and DOX-FNP2, the hydrodynamic diameters, measured by DLS analysis, were less than 200 nm, which was slightly increased compared to the blank FNPs shown in Table 5. The

morphology of the dispersed DOX-FNP complexes in the PBS buffer was also imaged using AFM and plotted in FIGs. 13C-D. As shown in FIGs. 13C-D, both DOX-FNP complexes were spheroidal nanoparticles with diameters of less than 200 nm, similar to the blank FNPs shown in FIG. 10. Table 6 also shows significant decrease in zeta potentials for the DOX-FNP complexes as compared to both the blank FNPs, indicating a direct association of DOX with the FNPs via electrostatic interactions (Wang et ah, J Biomed Nanotechnol 2013;doi: 10.1166/jbn.2013.1724). More importantly, the entrapment ratio of DOX in the FNPs could be as high as about 72%-77%, and the precipitated DOX-FNP complexes with such a high drug loading were demonstrated to be stable after the nano-sized DOX-FNP complexes were formed in the PBS buffer. As shown in FIG. 13B, the amount of DOX dissociated from the complexes in the PBS buffer was as low as about 20%. As such, the DOX- FNPs complexes dispersed in the PBS buffer were directly used as a nano-sized antitumor agent without further purification to remove free DOX for the subsequent studies, including uptake, cytotoxicity and immunochemotherapeutic effects.

[00119] Although similar chemical components were characterized for both FNPs in Table 5 and FIG. 11, showing that the polysaccharide including acidic GAG and neutral polysaccharides were main components in the FNPs, differences in chemical structures of both purified FNPs are believed to lead to different cytotoxicity of the FNPs and the DOX-FNP complexes, as well as the different physical properties including zeta potential and morphology (Tables 5 and 6, FIGs. 3 and 13).

[00120] In principle, pH-responsive release of DOX was expected from the nano- complexes formed via an electrostatic driving force between DOX and nanoparticles, which could provide a stimulus-responsive release mechanism after internalization by tumor cells or penetration into the tumor tissue in vivo (Chen et al., JPhys Chem B 2013; 117: 1261-8). The release profiles of DOX from both complexes at different pHs were further evaluated by immersing the dialysis tubes in large volume centrifuge tubes containing 6 ml of release buffers with different pH values. Specifically, 150 μΙ_, of the DOX-FNP1 complexes (168 μΜ for DOX), 200 μΐ, of DOX-FNP2 complexes (126 μΜ for DOX), or 84 μΙ_, of free DOX (300 μΜ) were placed in a dialysis tube (MWCO 300 K, Spectrum Labs, CA), and then immersed in tubes containing 6 ml of release buffers at different pH values (l xPBS, pH 7.4; 0.1 M acetic acid buffer, pH 5.5). All tubes were incubated at 37°C under mild agitation. The dialysate sample (0.5 mL) was collected at different time points and replenished immediately with the same volume of the fresh medium. The concentration of DOX in the dialysate was determined fluorometrically at 480 nm and 590 nm, and the cumulative release profiles were plotted verse release times.

[00121] As shown in FIG. 13E, a free DOX control confirmed that the dialysis membrane tubing with 300 K MWCO in this study could not restrict diffusion of the released drugs into the bulk release media in which the sink condition was

established, and they were able to reach 100% release after 5 hours. However, the release of DOX from both complexes at different pH values could not reach a plateau until at least 9-10 hours. The total released drug from both DOX-FNP complexes was significantly different under different pH conditions. Up to about 55% and about 65% of total drug were released at the physiological pH 7.4 for the DOX-FNP 1 and DOX- FNP2 complexes, respectively; however, around 80%> of total drug released at pH 5.5 were observed for both complexes. More importantly, the release rates of drug from both complexes increased with decreasing in the pH of release medium, indicating a pH-sensitive release behavior with accelerated release of DOX in an acidic

environment from both complexes. This favorable property is believed to facilitate passive tumor targeting and endosome escaping since the interstitial space of solid tumors and intracellular endosome compartments have a lower pH value (Wang et al., IntJPharm 2012;422:409-17).

Example 10: Cytotoxicity Studies of FNP Samples and DOX-FNP Complexes

[00122] The cytotoxicity of the purified FNP samples and the DOX-FNP complexes against four cancer cell lines (A549, B16BL6, MCF-7, and MCF-7/ADR cells) was evaluated by MTT assay (Wang et al., Int J Pharm 2012;422:409-17).

Biocompatibility of the purified FNP samples toward mouse fibroblast NIH3T3 cell was also measured through MTT assay (Liu et al, Biomaterials 2010;31 :5643-51). Briefly, 8000-10000 cells were plated in 96-well plates in 100 μΐ, culture media per well and incubated at 37°C in 5% C0 2 for 24 hours to allow the cells to attach.

Specifically, DMEM medium was used for A549 and B16BL6 cells, RPMI 1640 medium for MCF-7 and MCF-7/ADR cells, and DMEM-a medium for MIH3T3 cells. The media was supplemented with 10% fetal bovine serum (for tumor cells) or calf serum (only for NIH3T3), and 1.0 % penicillin-streptomycin. The cells were then treated with different concentrations of the FNPs or the DOX-FNP complexes. After a 48-hour treatment, 10 of MTT solution (5 mg/mL in PBS, pH 7.4) was then added to each well, and the plates were incubated for another 4 hours. The cell culture media was removed and replaced with 100 DMSO. The absorbance was measured by a microplate reader (Bio-Tek μ Quant) at 570 nm. For the DOX-FNP complexes, the average IC 50 value (the dose having 50% cell inhibition) was determined by cell survival plots using the "DoseResp" function in OriginPro 8.0. The results of studies were plotted in FIG. 14 and the IC 50 of the DOX-FNP2 complexes and free DOX are listed in Table 7.

Table 7

ICso (nM)

A549 B16BL6 MCF-7 MCF-7/ADR

DOX-FNP1 1170.60±92.33 494.87±38.00 1830.53±270.47 5464.57±16.87

DOX-FNP2 599.34±15.85 209.63±23.72 355.71±23.06 3522.55±110.03 Free DOX 1052.54±67.58 308.82±26.55 648.39±75.77 4177.36±116.12

[00123] The purified fractions, FNP1 and FNP2, both had mild to moderate cytotoxic activity against multiple tumor cell lines. However, compared to the FNP1, the FNP2 at the same GAG concentration had around 2-fold increase in cytotoxicity against the four tumor cell lines. There was synergistic cytotoxicity exerted by covalently conjugating DOX with FNPs via amide bond shown in Example 5. The DOX-FNP2 complexes showed significantly higher cytotoxicity against 4 tumor cells than free DOX after a 48-hour incubation. For A549, B16BL6 and MCF-7 cell lines, the IC 50 for the DOX-FNP2 complexes was about 1.5-1.8 fold lower than free DOX; even for the multidrug resistant cell line MCF-7/ADR, the IC 50 for the DOX-FNP2 was still about 1.2 fold lower than free DOX. These data demonstrates a synergistic cytotoxic effect when DOX was bound to one fraction of the FNPs, i.e., FNP2, even against the resistant tumor cell line. As shown in FIGs. 14G-J, at the respective IC 50 values of free DOX (Table 7), free DOX inhibited cell proliferation by 50%, whereas the DOX-FNP2 complexes showed about 62%-75% inhibition against the four tumor cells. According to a about 72% entrapment ratio for the DOX-FNP2 complexes (Table 7), the concentration of the FNP2 in the complexes at the respective IC 50 values of free DOX for the A549, B16B16 and MCF-7 cells was calculated to be less than 0.25 μg/mL of GAG concentration, and for the resistant MCF-7/ ADR cells less than 1.0 μg/mL of GAG concentration. FIGs. 14A-C shows almost no significant inhibition effect on the four tumor cells at that concentration for the FNP2, indicating that DOX and FNP2 in their physical complexes via the electrostatic interactions exerted synergistic cytotoxic effects and led to the IC 50 values 1.2-1.8 fold lower against the four types of tumor cells (Table 7).

[00124] Unexpectedly, the cytotoxicity of the DOX-FNP1 complexes was similar or even lower than free DOX in the four different cell lines, which is completely different from the DOX-FNP2 complexes. As shown in Table 7, the IC 50 values for the DOX-FNP1 complexes were about 1.1-2.8 fold higher than free DOX against 4 tumor cell lines, indicating that there is no obvious synergistic effect between DOX and the FNPl upon forming the physical complexes. The difference in the

cytotoxicity between the two DOX-FNP complexes was believed due to the different cytotoxicity of the FNPs, because the FNPl showed at least 2-fold higher cytotoxicity against different tumor cells than the FNP2 at the same GAG concentration as shown in FIGs. 14A-C. Presumably, the differences in the cytotoxicity of the FNPs portend different chemical structures for FNPl and FNP2 fractions, including polysaccharide chain, monosaccharide composites and linkages, uronic acid content, sulfation degree, and possible core proteins.

[00125] FIGs. 14A-C show cytotoxicity of the FNPs against four tumor cells, i.e., human non-small-cell lung cancer A549 cells, mouse melanoma B16BL6 cells, human breast cancer MCF-7 cells, and the multidrug resistant cell line MCF-7/ADR at different FNP concentrations. FIGs. 14D-F show cytotoxicity of the FNPs against mouse embryo fibroblast cell line NIH3T3 at different FNP concentrations. FIGs. 14G-J show cytotoxicity of the DOX-FNP complexes against four tumor cell lines (A549, B16BL6, MCF-7 and MCF-7/ ADR). The sigmoidal dose-response curves were fitted using OriginPro 8.0.

[00126] As shown in FIGs. 14A-C, the three FNP samples showed a dose- dependent cytotoxicity against the four tumor cell lines. For the crude FNP0, about 11-39% inhibition of cell proliferation were obtained in A549, B16BL6 and MCF-7 cells at the concentration ranging from 1-10 μg/mL. For the purified FNP2, the inhibition rates of 26-37% for B16BL6 cells, 9-33% for A549 cells, and 3-30% for MCF-7 cells were observed at the same concentration range. However, using a higher concentration (2-25 μg/mL), the purified FNP1 showed similar inhibition rates in the three tumor cells, i.e., 15-32% for B16BL6 cells, 11-28% for A549 cells, and 3-28% for MCF-7 cells. In comparison with the FNP1 and FNP2, similar inhibition rates found in the three tumor cells were resulted from different concentrations. The concentrations of the FNP1 were at least 2-fold higher than those of the FNP2, indicating that the purified FNP2 have stronger cytotoxic activity than the purified FNP1. In addition, it was observed that all the three FNP samples showed lower inhibition rates (8-18% for the FNP0, 5-15% for the FNP1, and 9-17% for the FNP2) in the multidrug resistant cell line MCF-7/ ADR. The lower inhibition rates here suggest that the FNPs only possess mild to moderate cytotoxic activity and the maximal concentration tested in this study is still not enough to effectively inhibit the proliferation of the resistant cells. Even for the sensitive tumor cells, the highest inhibition rates were still less than 40% at the maximal concentration of 10 μg/mL for FNP2 or 25 μg/mL for FNP1.

[00127] NIH3T3 is a mouse embryo fibroblast, which was commonly used in biocompatibility evaluation of nanomaterials. The mild to moderate cytotoxic activity of the FNPs were further evidenced by an in vitro biocompatibility test using NIH3T3 cell line as shown in FIGs. 14D-F. Less than 20% inhibition rates were seen in NIH3T3 cells treated with the three FNPs at the respective concentration ranges, suggesting that the FNPs did not have strong cytotoxic effect against normal cells, but had slightly higher cytotoxicity against tumor cells, especially towards the sensitive tumor cells. FIG. 15.

[00128] Even though the FNPs only possess mild to moderate cytotoxic activity, given that the FNPs induced the secretion of multiple proinflammatory cytokines and chemokines from immunocytes discussed above, it is believed that the FNPs are potential immunomodulators of biological responses in the adjuvant antitumor therapy in which the synergistic effect between the mild cytotoxic activity and the immunostimulatory activity is reached. For the purified FNPs, it has been

demonstrated that a similar immunostimulatory activity between the FNP1 and FNP2, inducing almost the same levels of cytokines and chemokines from immunocytes as shown in FIG. 12. However, as far as the cytotoxic activity concerns, the FNP2 showed around 2-fold stronger activity in the tumor cells tested in this study (FIGs. 14A-C).

Example 11: Apoptotic Assay and Cell Cycle Analysis of the FNPs

[00129] To better understand the difference in the cytotoxicity between the purified FNPs, the apoptotic effect and cell cycle arrest in the tumor cells treated by the purified FNPs were investigated. The apoptosis assay was conducted by evaluating DNA ladder formation. Briefly, A549 cells and B16BL6 cells were treated with the FNP samples at the GAG concentration of 10μg/ml, and then incubated at 37°C in 5% C0 2 for 48 hours. Apoptotic cells were then identified by TdT -mediated dUTP nick and labeling (TUNEL) assay using APO-BrdUTM TUNEL Assay Kit (Invitrogen, Eugene, OR) following the manufacturer's instructions. The cells were finally analyzed using flow cytometry (Epics XL Analyzer, Beckman Coulter Inc., Brea, CA) by collecting 20000 events for each sample and measuring the cell associated fluorescence. The results are plotted in FIGs. 15A-B.

[00130] To determine cell cycle distribution, A549 and B16BL6 cells were passed into 24-well plates and treated with the FNP solutions at the GAG concentration of 10 μg/mL, and then incubated at 37°C in 5% C0 2 for 24 hours. The cells were then trypsinized, washed with PBS, and fixed in 75% ethanol at 4°C for 2 hours. The fixed cells were stained with propidium iodide/RNase A staining buffer (Invitrogen, Eugene, OR) at 37°C for 30 min in the dark. The cell cycle analysis was conducted using flow cytometry (Epics XL Analyzer, Beckman Coulter Inc., Brea, CA) by collecting 20000 events for each sample and measuring the cell associated

fluorescence. The results are plotted in FIGs. 15C-D.

[00131] As shown in FIGs. 15A-B, the purified FNP2 induced strong apoptosis in the A549 cells and B16BL6 cells after an 48-hour incubation, and the crude FNPO had similar but weaker apoptosis induction in both tumor cells; however, the purified FNP1 could not induce significantly apoptotic effect in A549 tumor cells and induced only slight apoptotic effect in B16BL6 cells, which can explain the weaker cytotoxicity of the FNP1 as compared to the FNP2. The cell cycle arrest analysis using A549 and B16B16 cells treated with both purified FNPs substantially agrees with the apoptosis assay. As shown in FIGs. 15C-D, the FNP2 arrested the cell cycle at sub G0/G1 phase in both tumor cells after a 24-hour incubation, and the crude FNPO showed similar activities in both tumor cells. A significant increase in the sub G0/G1 peak, which corresponds to apoptotic cells, indicates that the tumor has undergone apoptosis. However, the purified FNP1 didn't significantly induce the cell cycle arrested at the sub G0/G1 phase, indicating different mechanisms for the cytotoxicity of the purified FNP1 and FNP2, although they showed similar immunostimulatory activity.

Example 12: Cellular Uptake and Confocal Microscopic Study of FNPs

[00132] In order to elucidate whether the different cytotoxicity between the DOX- FNP 1 and DOX-FNP2 complexes is related to DOX uptake and intracellular distribution after forming the complexes, the cell-associated DOX fluorescence intensity were quantitatively analyzed using flow cytometry after treatment of the tumor cell lines, A549 and B16BL6, with the two complexes. Quantification of intracellular DOX uptake in cancer cells was evaluated by flow cytometry. A549 and B16BL6 cells were cultured in 6-well plates at densities of l x lO 6 cells/mL, and incubated at 37°C in 5% C0 2 . The DOX-FNP complexes at the DOX concentration of 10 μΜ were added into each well, and free DOX was used as a control. After a 4-hour incubation, the media were aspirated. The cell monolayer was rinsed with PBS for three times, and then trypsinized. Flow cytometry analysis was carried out on a FACSCalibur (BD Biosciences) by collecting 20000 events for each sample and the data were analyzed by Flow Jo software (Tree Star, Ashland, OR). The results of the quantitative analyses were plotted in FIG. 16, DOX uptake by human non-small-cell lung cancer A549 cells (A-B) and mouse melanoma B16BL6 cells (C-D). As shown in FIG. 16, for both tumor cells, there was no significant difference in the DOX fluorescence for both the complexes and free DOX at the DOX concentration of 10 μΜ after a 4-hour incubation, indicating that DOX uptake wasn't impeded upon the formation of the DOX-FNP complexes via the electrostatic interactions. On the other hand, these data substantiate that the different cytotoxicity of both complexes was from their different cytotoxicity of the FNPs rather than the increase in the DOX uptake enhanced by the FNPs in the tumor cells.

[00133] To observe the internalization of the FNPs, the FNPs were first labeled with FITC (fluorescein isothiocyanate). Briefly, 3.5 mg/mL of FITC in DMSO was diluted to 0.7 mg/mL with 100 mM carbonate buffer (pH 9.3), and then 150 of the FNP1 (138.56 μg/ml) and FNP2 (56.83 μg/mL) were added into 400 μΐ. of the above carbonate buffer. After 24-hour incubation at the room temperature, resulting solution was dialysized (MWCO 300 K, Spectrum Labs, CA) against PBS buffer for three days. The cells were further treated with the FITC-labeled FNPs at the FITC concentration of 2 ng/mL, and the internalization of the FNPs was then imaged with confocal microscopy. To further observe the intracellular distribution of the two DOX-FNP complexes, nuclei and endolysosome were labeled with a nucleus-specific dye, Hoechst 33342 (blue), and an acidic endolysosome-specific dye, LysoTracker green DND-26, respectively. The cells were incubated with the samples at 37°C in 5% C0 2 for 4 hours, and then 100 nM Lysotracer Green DND-26 and 4 μΜ Hoechst 33342 were added for 30 min incubation prior to imaging by the confocal microscopy.

[00134] Confocal laser scanning microscopy (FluoView FV1000, Olympus, Japan) was used to investigate intracellular DOX distribution in tumor cells treated with the DOX-FNP complexes. Briefly, the cells were seeded on cover slips with a density of 10 6 cells/mL in 6-well plates and cultured at 37°C in 5% C0 2 for 24 hours. The cells were then treated with the DOX-FNP complexes at the DOX concentration of 10 μΜ for 4 hours. Free DOX was used as a control. To observe the intracellular distribution, endolysosome- and nuclear-specific markers, LYSOTRACKER™ green (100 nm) and Hoechst 33342 (4 μΜ), were incubated with the cells for 30 min prior to the confocal imaging. After that, the cover slip was washed with PBS three times, set on a microscope slide, and then examined by confocal microscopy. The FNP1 and FNP2 were confirmed to be efficiently taken up by both tumor cells after a 4-hour incubation. The internalization of FNPs themselves by tumor cells substantially indicates that the FNPs could mediate the uptake and distribution of DOX in tumor cells via the DOX-FNP complexes, instead of free DOX released from the complexes.

[00135] Upon confirming the uptake of the FNPs by tumor cells described above, sub-cellular distribution of the DOX-FNP complexes in both A549 and B16BL6 tumor cells were imaged. As shown in FIG. 17, the confocal analysis showed that different intracellular distribution of both DOX-FNP complexes and free DOX was observed in both tumor cells (in human non-small-cell lung cancer A549 cells (A) and mouse melanoma B16BL6 cells (B)) after a 4-hour treatment. Scale bars represent 10 μιη. Majority of DOX in both cells incubated with both complexes were

predominantly distributed in the endolysosomal compartment, while most of the free DOX was located outside the organelle. Major nanoparticles which were internalized via endocytosis in mammalian cells were mainly found within endosomes or lysosomes (Iversen et ah, Nano Today 2011;6:176-85). Thus, the DOX-FNP complexes appeared to be internalized by endocytic pathway in both the tumor cell lines.

[00136] In this study, the DOX-FNP complexes were demonstrated to have a pH- sensitive release behavior with accelerated release of DOX in an acidic environment a shown in FIG. 13E, which facilitate DOX escaping from endosome or lysosomes after internalization of both complexes in tumor cells. Hence, the different distribution is not another cause that led to the different cytotoxicity for both DOX-FNP complexes. Overall, the experimental data here indicate that even though there was a different sub-cellular distribution, the formation of the DOX-FNP complexes did not decrease DOX uptake by both tumor cell lines, which further supports that different cytotoxicity of both complexes was from their different cytotoxicity of the FNPs themselves against tumor cells.

Example 13: Co-culture System to Evaluate Immunochemotherapeutic Activity

[00137] A co-culture system using B 16BL6 tumor cells and the splenocytes derived from C57BL/6 mice was used to evaluate the immunotherapeutic effect of the DOX-FNP complexes in vitro. Briefly, 2 l0 5 tumor cells, labeled with 5 μΜ CFSE, were co-cultured with 5 xlO 6 splenocytes, and then the co-cultures were treated with free DOX and both DOX-FNP complexes at the DOX concentration of 1 μΜ. After 24-hour incubation, the death of tumor cells was determined by the PI uptake method using flow cytometer after gating on the CFSE labeled cancer cells.

[00138] After demonstrating differences in cytotoxicity for both DOX-FNP complexes against tumor cells, the idea to combined immunochemotherapy using the complexes was further confirmed by the co-culture analysis. The co-culture study is an in vitro model system by mimicking in vivo situation (Roy et ah, Pharm Res 2012;29:2294-309; Roy et al, Mo I Pharm 2010;7: 1778-88). For such a purpose, B16BL6 tumor cells were first labeled with CFSE, and co-incubated with splenocytes isolated from C57BL/6 mice. The co-cultures were then incubated with either DOX- FNP complexes, or free DOX at the DOX concentration of 1 μΜ for 24 hours. The death of the tumor cells was determined by the PI uptake method using flow cytometry after gating on the CFSE labeled cancer cells. The untreated co-cultured cells were used as a negative control. Significantly higher death of the cancer cells was observed with the treatment of the DOX-FNP2 complexes as compared to free DOX or the DOX-FNPl complexes as shown in FIG. 18, which is consistent with the data in the direct cytotoxicity experiment using MTT assay as shown in FIGs. 14G-J and Table 7. As shown in FIGs. 18A-D, results from co-cultures treated with the negative control (A), free DOX (B) DOX-FNPl complex (C), and DOX-FNP2 complexe (D) at the DOX concentration of 1 μΜ are plotted. Statistical analysis of the mean DOX fluorescence associated with the cells was performed and significant differences (P<0.05) between different treatments were observed and plotted in FIG. 18E. As the DOX-FNP2 complexes had both the cytotoxic and immunostimulating activities, DOX and FNP2 are believed to work with each other to produce a synergistic effect, resulting in a higher death rate in the co-culture cells treated with the complexes. Compared to the direct cytotoxicity experiment where the DOX-FNPl complexes had lower cytotoxicity than free DOX as shown in FIGs. 14G-J and Table 7, in the co-culture experiment where a mixed culture of cancer cells and splenocytes were treated, the DOX-FNPl complexes unexpectedly enhanced tumor cell death as compared to free DOX alone. In this co-culture experiment, a higher death rate of the cancer cells exerted by both complexes, especially the DOX-FNPl complexes is attributed to the immune stimulatory activity of the FNPs, as both FNPs have been shown to induce the secretion of multiple pro-antitumor cytokines and chemokines from splenocytes, such as TNF-a and MIP-la, which has direct cytotoxic activity. Overall, the enhanced tumor cell death by both complexes in this experiment confirmed the potential to use the DOX-FNP complexes in vivo for combined cancer immunochemotherapy.

[00139] The materials and methods of the appended claims are not limited in scope by the specific materials and methods described herein, which are intended as illustrations of a few aspects of the claims and any materials and methods that are functionally equivalent are within the scope of this disclosure. Various modifications of the materials and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative materials, methods, and aspects of these materials and methods are specifically described, other materials and methods and combinations of various features of the materials and methods are intended to fall within the scope of the appended claims, even if not specifically recited. Thus a combination of steps, elements, components, or constituents can be explicitly mentioned herein; however, all other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.