NANO-PARTICULATE COMPOSITIONS FOR STIMULATING HOST INNATE IMMUNE RESPONSES FOR THERAPEUTIC APPLICATIONS

Abstract

Novel biocompatible Fenton-catalytic nano-particulate composites preferably based on nanoparticle (NP)-based catalysts, one or more reducing agents, and one or more peroxide compounds are formulated to take advantage of their ability to stimulate bactericidal as well as anti-tumor immune response by means of eliciting the generation of reactive oxygen species (ROS) in immune cells, in particular, in macrophages. The therapeutic composition can serve as a treatment for wound infections by, but not limited to, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Klebsiella pneumoniae, and Acinetobacter baumannii, as a wound/lesion dressing that provide an anti-bacterial immune environment for the accelerated wound healing. In a similar principle, the therapeutic composition can serve as a treatment for solid tumors by providing an anti-tumor immune environment that inhibits tumor growth.

Claims

1. A Fenton-catalytic nano-particulate composite comprising: (a) one or more nanoparticle-based catalyst comprising Fe.sub.xA.sub.1-xFe.sub.2O.sub.4 (0≤x≤1) nanoparticles wherein A is Mg, Mn, Zn, Cu, Cr, Co, or Ni, or any combination of said different nanoparticles, and wherein, independently, said nanoparticles have a particle size of from about 2 nm to about 500 nm at a concentration of from about 0.1 to about 100 milli grams per milli liter; (b) including one or more reducing agents, and (c) including one or more peroxide compounds.

2. The composition of claim 1, wherein the amount of said one or more reducing agents is from about 3 μM to about 300 mM.

3. The composition of claim 2, wherein the amount of said one or more peroxides is from about 3 μM to about 1,000 μM.

4. The composition of claim 3, wherein said one or more reducing agents comprise (i) vitamin C, (ii) vitamin E, (iii) erythorbic acid (iv) glutathione, (v) citric acid, (vi) pyruvic acid, (vii) lactic acid, (viii) glucose, and (ix) erythrose, or any combination thereof.

5. The composition of claim 4, wherein said one or more peroxides comprise (i) hydrogen peroxide, (ii) benzoyl peroxide, (iii) acetyl benzoyl peroxide (acetozone), and (iv) artemisinin or any derivative thereof, or any combination thereof.

6. The composition of claim 5, wherein the amount of said one or more nanoparticles is from about 1 to about 5 milli grams per milli liter; wherein the size of said one or more nanoparticles is from about 3 nm to about 120 nm; and wherein the amount of said one or more reducing agent is from about 500 μM to about 1.5 mM.

7. The composition of claim 6, wherein the amount of said one more peroxides is from about 100 μM to about 500 μM.

8. The composition of claim 7, wherein said reducing agent comprises erythorbic acid.

9. The composition of claim 8, wherein said peroxide comprises artemisinin or a derivative thereof.

10. An aqueous solution comprising the composition of claim 1.

11. A hydrogel comprising the composition of claim 1.

12. A liposome comprising the composition of claim 1.

13. An aqueous solution comprising the composition of claim 9.

14. A hydrogel comprising the composition of claim 9.

15. A liposome comprising the composition of claim 9.

16. A method for treating infected wounds or lesions comprising the step of applying the composition of claim 1 to a wound or lesion.

17. A method of treating a solid tumor comprising the step of applying a local injection of the composition of claim 1 to said solid tumor.

18. The method of claim 16, wherein said infection comprises Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Klebsiella pneumoniae, and Acinetobacter baumannii, or any combination thereof.

19. The method of claim 17, wherein said tumor comprises breast cancer, lung adenocarcinoma, cervical cancer, ovarian cancer, prostate cancer, melanoma, or renal cell carcinoma, or any combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 shows the effects of Fe.sub.3O.sub.4 iron oxide NPs (IONPs) on the generation of ROS and the on the bactericidal activity in RAW 264.7 macrophages against intracellular S. aureus. A. The quantification of ROS generation in the RAW 264.7 cells treated with varying concentration of IONPs (0-3 mg/mL). B. The colony number of viable bacteria (S. aureus) in the lysed RAW 264.7 macrophages treated with varying concentration of IONPs (0-3 mg/mL) following the exposure of live S. aureus.

[0012] FIG. 2 shows the synergistic effects of reducing agent, vitamin C (VC), and hydrogen peroxide (H.sub.2O.sub.2) on the IONPs-mediated generation of ROS and bactericidal activity in RAW 264.7 macrophages against intracellular S. aureus. (A-B) The quantification of ROS generation (A) and viable colony number of S. aureus (B) in the RAW 264.7 cells treated with VC (500 μM) alone, IONP (3 mg/mL) alone, or IONP (3 mg/mL)+VC (500 μM). (C-D) The quantification of ROS generation (C) and viable colony number of S. aureus (D) in the RAW 264.7 cells treated with IONP (3 mg/mL) alone, or IONP (3 mg/mL)+H.sub.2O.sub.2 (100 μM). E. Representative images of surviving S. aureus colony on agar plates from lysed RAW264.7 macrophages treated with H.sub.2O.sub.2 (100 μM), IONP (3 mg/mL), or IONP+H.sub.2O.sub.2. N=3-5 per group, *p<0.05

[0013] FIG. 3 shows the synergistic effects of IONP and vitamin C on triggering a Fenton reaction that generates hydroxyl radicals in RAW264.7 macrophages. A. The intracellular level of ferrous iron (Fe2+) in RAW 264.7 cells treated with IONPs (3 mg/mL) alone, IONPs with VC (500 μM), or IONPs with VC and BIP (iron chelator, 500 μM). B. The intracellular level of hydroxyl radical concentration in RAW 264.7 cells treated with IONPs (3 mg/mL) alone, IONPs with VC (500 μM), or in combination with BIP (500 μM) for 24 h. C. The bactericidal activity of RAW 264.7 cells treated with IONPs (3 mg/mL) with VC (500 μM), IONPs with VC and BIP (500 μM), or IONPs with VC and TIH (200 mM), assessed by antibiotic protection assay. *p<0.05, N=3-5 per group.

[0014] FIG. 4 shows the in vivo validation of the efficacy of IONPs, alone or in combination with reducing agent (VC) in the mouse model of wound S. aureus infection. A. The quantification of bacterial CFU number from wounds of C57BL/6 mice. *p<0.05, N=6 per group. S. aureus (1×10.sup.6 CFU/wound) was inoculated to 6-mm skin wounds at 0 day followed by topical application of IONPs or IONPsNC on each wound at 1 day. The skin wound tissues were dissected at 2 day for bacteria CFU counting and qPCR analysis. B. The expression of M1 marker (iNos and II-1β) and M2 marker (Arg-1 and Cd206) in the F4/80+ macrophages isolated from wounds mice treated with IONPs or IONPs with VC. *p<0.05, N=4 per group.

[0015] FIG. 5 shows a schematic on the proposed mechanism by which Fenton-catalytic nano-particulate composites can promote the killing of intracellular bacteria via triggering a Fenton reaction that generates intracellular ROS, in particular, hydroxyl radicals (OH.sup.−).

[0016] FIG. 6 shows a schematic of a liposome encapsulating the three ingredients of Fenton-catalytic nano-particulate composites.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The current invention comprises a composition containing a pharmaceutical or medical grade NP catalyst, reducing agent, and peroxide in an aqueous vehicle or hydrogel.

[0018] The one or more active NP catalyst contains nanoparticles E. G. Fe.sub.xA.sub.1-xFe.sub.2O.sub.4(O≤x≤1) where A is Cr.sup.2+, Co.sup.2+, or Ni.sup.2+, or one of the following solid solutions with the particles size in the size range of from about 2 nm to about 500 nm at concentrations between from about 0.1 mg/mL-100 mg/mL: (i) Fe.sub.xMg.sub.1-xFe.sub.2O.sub.4 (0≤x≤1), (ii) Fe.sub.xCu.sub.1-xFe.sub.2O.sub.4 (0≤x≤1), (iii) Fe.sub.xMn.sub.1-xFe.sub.2O.sub.4 (0≤x≤1), and (iv) Fe.sub.xZn.sub.1-xFe.sub.2O.sub.4 (0≤x≤1). Based on our cell culture experiment, the desirably concentration of NP catalyst is from about 1 to about 5 mg/mL and a size of from about 3 nm to about 120 nm., and preferably from about 4 nm to about 20 nm

[0019] The one or more reducing agents contain single or a combination of two or more the following reducing agents: (i) vitamin C, (ii) vitamin E, (iii) erythorbic acid (iv) glutathione, (v) citric acid, (vi) pyruvic acid, (vii) lactic acid, (viii) glucose, and (ix) erythrose, at concentrations in the range of 3 IM to 300 mM. Based on our cell culture experiment, the preferable concentration of reducing agent is from about 500 μM to about 1.5 mM. Among all these reducing agents, vitamin C and erythorbic acid exhibit the stronger reducing effect than the other. However: erythorbic acid is a synthetic stereoisomer of ascorbic acid and widely used as an antioxidant in processed foods. As such, the rate of metabolism for erythorbic acid in the human body is slower than that for vitamin C, which provides longer lasting reducing action. Hence, erythorbic acid is the preferred choice for this invention.

[0020] The one or more peroxide compound may be a single or a combination of two or more the following peroxo-containing agents: (i) hydrogen peroxide, (ii) benzoyl peroxide, (iii) acetyl benzoyl peroxide (acetozone), and (iv) artemisinin and derivatives thereof, and any combination thereof. The latter all contain an endoperoxide ring that makes the peroxo functional group much more stable than the normal open-chain peroxide compounds, and hence is the preferred choice for this invention. Furthermore, the concentration of peroxide compound is in the range of from about 3 μM to about 1,000 μM. Based on our cell culture experiment, the desired concentration of hydrogen peroxide is from about 100 μM to about 500 μM, and preferably from about 100 μM to about 300 mM.

[0021] The proof of principle for the application of Fenton-catalytic nanocomposite for treating bacterial infection was validated using in vitro culture models of macrophage-like RAW 264.7 cells and in vivo mouse model of skin wound infections by S. aureus, wherein Fe.sub.3O.sub.4 iron-oxide NP (IONP, 100 nm) and vitamin C (VC) were used as a NP catalyst and reducing agent, respectively. Then, we have examined if IONPs, alone or in combination with a VC or hydrogen peroxide, can be beneficial for macrophage-mediated bactericidal and pro-inflammatory immune responses against S. aureus.

[0022] Once IONPs are internalized by macrophages, IONPs are degraded in endocytic organelles, resulting in the release of iron ion (Fe.sup.3+) in the cytoplasm. The newly formed iron ions can considerably affect the intracellular redox signaling that leads to the generation of ROS inside cells via a Fenton reaction. Thus, we investigated whether a IONPs-triggered Fenton reaction to generate ROS is sufficient to exhibit a bactericidal activity against Gram-positive bacteria, S. aureus, survived within macrophages. To ascertain this, we have assessed the capacity of IONPs to produce ROS in RAW 264.7 cells by treating the cells with varying concentrations of IONPs (0-3 mg/mL) and then quantifying the extent of total ROS generation using carboxy-H.sub.2DCFDA, fluorogenic dye that can detect hydroxyl, peroxyl and other ROS activity within the cell. The levels of intracellular ROS in RAW 264.7 cells in response to IONPs were increased in a dose dependent manner (FIG. 1A). Importantly, the number of viable intracellular S. aureus was decreased with increasing concentrations of IONPs (FIG. 1B), which support that IONPs are capable of eliciting a bactericidal function of macrophages against intracellular S. aureus to some extent and this is associated with the capacity of IONPs to trigger the generation of ROS in macrophages.

[0023] Since the ability to increase the availability of ferrous iron (Fe.sup.2+) in the cytoplasm is critical for ROS formation, the effect of reducing agents (VC) on the generation of ROS and bactericidal activity of macrophages was tested in macrophages. The combined treatment of IONPs with VC (500 μM) to RAW 264.7 macrophages could synergistically augment the generation of ROS (FIG. 2A), which was associated with increased bactericidal activity (FIG. 2B). Since the oxidation of Fe.sup.2+ by hydrogen peroxide (H.sub.2O.sub.2) produces highly reactive hydroxyl radical, the effect of hydrogen peroxide on the generation of ROS and bactericidal activity of macrophages was tested as well. Similar to the case of VC, the combined treatment of IONPs with VC (500 μM) to RAW 264.7 macrophages could significantly augment the generation of ROS (FIG. 2C), which was associated with increased bactericidal activity (FIGS. 2D and 2E).

[0024] To further determine if the VC-mediated ROS generation and bactericidal activity were associated with a Fenton reaction due to increased release of Fe.sup.2+, the levels of Fe.sup.2+ were compared between RAW 264.7 cells treated with IONPs alone and IONPs with VC. The treatment of IONPs alone could significantly increase the level of Fe.sup.2+ in RAW 264.7 cells by 3-fold compared to the untreated cells, and its level was further augmented by 2-fold in the presence of VC, compared to IONPs only (FIG. 3A). Among various forms of ROS, hydroxyl radical (OH.sup.−) is highly cytotoxic by causing oxidative damage to DNA and cell membrane. In the Fenton reaction-mediated generation of ROS, the oxidation of Fe.sup.2+ by hydrogen peroxide (H.sub.2O.sub.2) produces highly reactive hydroxyl radical. To test if enhanced bactericidal activity of RAW264.7 cells with IONPs in combination with VC could be a consequence of Fe.sup.2+ release and the generation of hydroxyl radicals, the extent of hydroxyl radical generation and bactericidal activity in the RAW 264.7 cells treated with IONP and VC were quantified using the chelator of Fe.sup.2+, BIP, or scavenger of hydroxyl radicals, thiourea (THI) The treatment of BIP to RAW 264.7 cells significantly decreased IONPs and VC-induced generation of hydroxyl radicals up to the level of IONPs treatment only (FIG. 3B), which was associated with a decrease in bactericidal activity of RAW 264.7 cells (FIG. 3C). Additionally, the inhibition of hydroxyl radical formation by BIP was sufficient to reverse IONPs/VC-induced bactericidal activity of RAW 264.7 cells, which was comparable to the level induced by THI treatment. Taken together, these results support the synergistic effect of IONPs and VC in triggering a Fenton-like reaction in the macrophages, which contributed to the killing of intracellular bacteria.

[0025] By observing the capacity of IONPs, in combination with VC, in promoting the bactericidal activity of RAW 264.7 cells in vitro, its efficacy was validated in vivo using a murine model of wound infection by S. aureus. The viable number of S. aureus was quantified from the wounded skin harvested at day 2 post-infection (FIG. 4A). The treatment of IONPs to the wound could reduce a bacterial burden by 25% compared to the control group (p<0.05). In consistence with our results from the in vitro study, the co-treatment of VC (500 μM) with IONPs significantly reduced S. aureus numbers in the wound by 75% compared to the control group. The F4/80.sup.+ macrophages from wounds of mice treated with IONPs and VC exhibited a significantly increased expression of iNos and II-1β compared to either IONPs alone or the untreated control group, which was associated with an attenuated expression of M2 markers including Arg-1 and Cd206 (FIG. 4B). Taken together, our results support that Fenton-catalytic nano-particulate composites can promote a bactericidal activity of macrophages by triggering a Fenton-like reaction (FIG. 5).

[0026] To improve the topical or intravenous delivery of the biocompatible Fenton-catalytic nano-particulate composites, a liposome encapsulating the three ingredients can be used as a drug-carrying vehicle to administrate the drug. A liposome is a spherical vesicle consisting of single or multiple lipid bilayers of phospholipids, for example, phosphatidylcholine or egg phosphatidylethanolamine. The aqueous solution core of a properly prepared liposome is surrounded by a hydrophobic lipid bilayer. Such structure allows the water-soluble nanoparticle-based Fenton catalyst and the reducing agent to be encapsulated in hydrophilic core, and on the other hand, the oil-soluble peroxide compound to be encapsulated in lipid bilayer (FIG. 6). The lipid bilayer to fuse with the biological cell membrane to enhance drug delivering efficiency. Such liposomes can be readily prepared by the thin-film hydration method followed by sequential extrusion. The following procedure is provided as an example for the encapsulation of the biocompatible Fenton-catalytic nano-particulate composites: a 50-mL chloroform solution containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (Chol) in the mass ratio of 5:1 (the total lipid weight=10 mg) is added into a round-bottomed flask and heated at 50° C. in a water bath to remove the chloroform form the lipid film by a rotary evaporator. The film formed at the bottom of the flask is further evaporated under vacuum for 12 hours remove residual chloroform. The dry film was then hydrated by adding 1 mL of aqueous solution containing iron oxide nanoparticles (15 mg), vitamin C (30 mg) and artemisinin (15 mg) at 50° C. water bath for 30 min to produce liposomes loaded up with nano-particulate composites. The liposome dispersion is then homogenized with a mini extruder at 50° C. through a polycarbonate filter (average pore size=200 nm) for 20 times. Non-encapsulated iron oxide nanoparticles, vitamin C and artemisinin are removed by dialysis in a membrane dialysis bag.

[0027] While in accordance with the Patent Statutes, the best mode and preferred embodiments have been set forth, the scope of the invention is not limited thereto, but rather, by the scope of the attached claims.