Method for promoting wound healing.

Abstract

A method of promoting wound healing in a patient, the method comprising applying on a wound a biodegradable amino-acid based polymer.

Claims

1. A method of promoting wound healing in a patient, the method comprising: applying on the wound a composition including an amino-acid based polymer, wherein the amino-acid based polymer is selected from (1) a poly (ester amide urea) wherein at least one diol, at least one diacid, and at least one amino acid are linked together through an ester bond, an amide bond, and a urea bond, (2) a poly (ester urethane urea) wherein at least one diol and at least one amino acid are linked together through an ester bond, a urethane bond, and a urea bond, (3) a poly (ester amide urethane urea) wherein at least one diol, at least one diacid, and at least one amino acid are linked together through an ester bond, an amide bond, a urethane bond, and a urea bond, (4) a poly (ester amide urethane) wherein at least one diol, at least one diacid, and at least one amino acid are linked together through an ester bond, an amide bond, and a urethane bond, (5) a poly (ester urea) wherein at least one diol and at least one amino acid are linked together through an ester bond and a urea bond, and (6) a poly (ester urethane) wherein at least one diol and at least one amino acid are linked together through an ester bond and a urethane bond, further wherein the at least one diol is a compound of formula: HO—R.sub.1—OH, R.sub.1 is chosen from C.sub.2-C.sub.12 alkylene optionally interrupted by at least one oxygen, C.sub.3-C.sub.8 cycloalkylene, C.sub.3-C.sub.10 cycloalkylalkylene, ##STR00015## the at least one diacid is a compound of formula: HO—(CO)—R.sub.3—(CO)—OH, R.sub.3 is C.sub.2-C.sub.12 alkylene, the at least one amino acid is chosen from naturally occurring amino acids and non-naturally occurring amino acid.

2. The method as defined in claim 1, wherein the amino-acid based polymer is a poly (ester amide urea) comprising the following two blocks with random distribution thereof: ##STR00016## wherein the ratio of l:m ranges from 0.05:0.95 to 0.95:0.05, l+m=1, R.sub.1 is chosen from C.sub.2-C.sub.12 alkylenes optionally interrupted by at least one oxygen, C.sub.3-C.sub.8 cycloalkylenes, C.sub.3-C.sub.10 cycloalkylalkylenes, ##STR00017## R.sub.3 is C.sub.2-C.sub.12 alkylene, R.sub.2 and R.sub.4 are independently chosen from the side chains of L- and D-amino acids so that the carbon to which R.sub.2 or R.sub.4 is attached has L or D chirality.

3. The method as defined in claim 1, wherein the polymer is in the form of polymer microcapsules.

4. The method as defined in claim 1, wherein the polymer is in the form of polymer nanocapsules.

5. The method as defined in claim 3, wherein the microcapsules are suspended in a liquid.

6. The method as defined in claim 1, wherein the composition is sprayed on the wound.

7. The method as defined in claim 1, wherein the biodegradable amino-acid based polymer is devoid of anti-bacterial agents.

8. The method as defined in claim 1, wherein the biodegradable amino-acid based polymer is devoid of bacteriophages.

9. The method as defined in claim 1, wherein the biodegradable amino-acid based polymer is essentially devoid of bacteriophages.

10. The method as defined in claim 1, wherein the polymer is in the form of a film.

11. The method as defined in claim 1, wherein the polymer is applied repeatedly on the wound.

12. The method as defined in claim 11, wherein the polymer is applied daily on the wound.

13. The method as defined in claim 12, wherein the polymer is applied until the wound reaches a predetermined healing status.

14. The method as defined in claim 1, wherein the wound is a chronic wound.

15. The method as defined in claim 14, wherein the chronic wound is selected from the group consisting of diabetic foot ulcers, pressure ulcers, venous stasis ulcers, and ischemic ulcers.

16. The method as defined in claim 1, wherein the composition further comprises at least one of an analgesic and an anti-inflamatory agent.

17. The method as defined in claim 1, wherein the composition further comprises at least one of ibuprofen, lidocaine, opioids, and cannabinoids.

18. The method as defined in claim 3, wherein the microcapsules consist essentially of the amino-acid based polymer.

19. The method as defined in claim 3, wherein the microcapsules consist of the amino-acid based polymer.

20. The method as defined in claim 4, wherein the nanocapsules consist essentially of the amino-acid based polymer.

21. The method as defined in claim 4, further comprising a permeation enhancer promoting permeation of the nanocapsules in the wound.

22. The method as defined in claim 1, wherein the composition further includes at least one additional component selected from the group consisting of: metal ions, zinc ions, silver ions, anti-microbial peptides (AMPs), Human Cathelicidin LL-37, Innate Defense Regulator 1018 peptide (IDR-1018), Human β-defensis (hBD-2 and hBD-3), Pexiganan, Tiger17 peptide, Growth factors (Gfs), granulocyte-macrophage colony-stimulating factor (GM-CSF), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), stem cells, bone marrow-derived stem cells (BMSCs), adipose-derived stem cells (ADSCs), cellular adhesion molecules (CAMs), antibiotics or antimicrobials, analgesics, polyethylene glycol and anti-inflammatory agents.

23. The method as defined in claim 1, wherein the composition is part of a dressing selected from the group consisting of gauzes, transparent films, foams, hydrogels, hydrocolloids, and hydroconductive dressings.

24. The method as defined in claim 1, wherein the composition is dispersed in or covalently bonded to the remainder of the dressing.

25. The method as defined in claim 1, further comprising degrading the composition in tissues part of the wound or adjacent to the wound.

26. The method as defined in claim 1, wherein the composition is in the form of a coating layer coats an implantable device.

27. The method as defined in claim 26, wherein the coating layer is designed to completely biodegrade over of predetermined duration, the predetermined duration being one of at least one month and at least one year.

28. The method as defined in claim 1, wherein where R.sub.1 is —(CH.sub.2).sub.6—, R.sub.3 is —(CH.sub.2).sub.8—, and R.sub.2 and R.sub.4 are the side chain of L-leucine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] In the drawings:

[0076] FIG. 1, in scanning microscope micrographs, illustrates degradation of PEAU microcapsules by various agents;

[0077] FIG. 2, in photographs, illustrates typical result of treatment of non-infected wounds in placebo (top) and BACTELIDE treated (bottom) rats;

[0078] FIG. 3, in a histological H&E stain, illustrates a wound treated with BACTELIDE (magnification 20×);

[0079] FIG. 4 is a halftoned version of FIG. 3; and

[0080] FIG. 5 illustrates particle size distribution in a microcapsule formulation usable to perform the proposed method.

DETAILED DESCRIPTION

[0081] The examples below use a polymer referred to as PEAU. This polymer is a poly (ester amide urea) comprising the following two blocks with random distribution thereof:

##STR00013##

wherein
the ratio of l:m ranges from 0.05:0.95 to 0.95:0.05, l+m=1,
R.sub.1 is chosen from C.sub.2-C.sub.12 alkylenes optionally interrupted by at least one oxygen, C.sub.3-C.sub.8 cycloalkylenes, C.sub.3-C.sub.10 cycloalkylalkylenes,

##STR00014##

R.sub.3 is C.sub.2-C.sub.12 alkylene,
R.sub.2 and R.sub.4 are independently chosen from the side chains of L- and D-amino acids so that the carbon to which R.sub.2 or R.sub.4 is attached has L or D chirality.

[0082] The more specific polymer referred to in the examples is the polymer wherein R.sub.1 is —(CH.sub.2).sub.6—, R.sub.3 is —(CH.sub.2).sub.8— and both R.sub.2 and R.sub.4 are the side chain of L-leucine. These polymers are referred to hereinbelow in abbreviated form as (8L6).sub.l-(1L6).sub.m.

[0083] The amino-acid based polymers and microcapsules may be prepared as follows, although other preparation methods are within the scope of the invention. PEAU is synthesized, in some embodiments, by interfacial polycondensation of the monomer L6, di-p-sulfonic acid salt of bis-(L-leucine)-1,6-hexylene diester with trisphogene/sebacoyl chloride with water/dichloromethane system. This method is fast, irreversible, involves two immiscible phases at room temperature and lead to high molecular weight polymer. Synthesis of the monomer L6 can be executed in the presence of p-toluene sulfonic acid by condensation of L-leucine with 1,6-hexanediol in refluxed cyclohexane. Purification includes recrystallization from water, filtration and drying under vacuum. The compositions containing microcapsules are fabricated using a water-in-oil-in-water double emulsion-solvent, where the addition of the bacteriophages occurs in some embodiments in the secondary emulsion to minimize their exposure with the solvent dichloromethane (DCM). The DCM can also be replaced by an other suitable solvent, such as ethyl acetate, chloroform, or another organic solvent. Polymer films may be manufacturing by spraying the polymer solution and drying. Powders may be obtained by grinding films or microcapsules, among other possibilities.

[0084] While evaluating in-vivo toxicity of the PEAU polymer, it was discovered that, unexpectedly, the PEAU polymer promoted wound healing. Indeed, application of a PEAU polymer formulation including bacteriophages led to wounds that healed faster than a control, even in sterile conditions in which the bacteriophages are expected to have no influence. It is also expected that the other amino-acid based polymers described in the present document would have similar effects based on their similar chemistry. Accordingly, using these polymers for wound treatment, even in the absence of bacteriophages would promote faster healing compared to the current standard of care.

[0085] More specifically, a study included a total of 44 rats, 40 of which were selected for test or control article treatment and randomized into groups based on weight and sex. Animals were microchipped for body temperature monitoring. One uniform size full-thickness excisional wound was created using a sterile template (16 mm diameter) and a surgical blade at the lateral/dorsal region. Baseline wound measurements and temperatures were taken. A predetermined volume of test (referred to as BACTELIDE herein, the composition of which is detailed below) or control (physiological buffer) (230-250 mL) was applied to the wound site by one actuation of a spray bottle pump. The spray was allowed to sit undisturbed for approximately five minutes. The site was covered with Mepilex® Lite foam dressing, and a secondary non-occlusive dressing. After a 24±2 hours exposure, the wound was measured and the treatment was reapplied. The application or re-application and wound measurement were performed for 28 consecutive days beginning on Day 1. At the Day 29 timepoint 20 animals had blood collected and then were euthanized. Tissues were collected, with a select list processed for histopathology evaluation. After a two-week recovery phase, blood was collected and animals were euthanized. Tissues were collected, with a select list processed for histopathology evaluation.

[0086] The BACTELIDE composition included a mixture of 14 lytic bacteriophages. These phages were encapsulated into a biodegradable polyester amide) urea (PEAU) co-polymer in the form of microcapsules, and delivered by a dosage-metered spray bottle and pump. Bactelide includes the microcapsules in suspension in a mixture including 17.33 mg/mL PEAU, 10 mg/mL PVA, 0.5844 mg/m: NaCl: 12037 MgSO.sub.4, and 0.6057 mg/mL TRIS HCl.

[0087] There were no adverse findings in the histopathology data of the skin or axillary lymph nodes attributed to BACTELIDE. The findings present were as expected following surgical wound creation and wound treatment. Under the conditions of this full-thickness wound-healing study and based on the Irritant Rank Score, BACTELIDE was considered a non-irritant when compared with the control (PBS only) at day 29 and day 43±1, and a moderate irritant compared to untreated skin at day 29 and day 43±1. The moderate irritant status compared to untreated skin was as expected, because the untreated skin contained no foreign material and had not received a surgical wound, and so only exhibited background findings. Wounds of male and female animals enrolled in BACTELIDE groups were fully healed prior to the wounds of male and female animals enrolled in control groups. All control male animals were fully healed by day 23 as compared to 18 days in the males treated with BACTELIDE; all female animals in the control group were fully healed by day 20 as compared to day 15 in the BACTELIDE. Rate of wound healing in the BACTELIDE test group was significantly higher 16.88% faster healing on day 7 and reaching 19.18% faster healing on day 10 for all groups combined. FIG. 2 illustrates typical result of treatment of non-infected wounds in placebo (top) and BACTELIDE treated (bottom) rats. Faster healing is clearly shown (middle column), as well as reduction in scar visibility (last column).

[0088] It is hypothesized that PEAU contributes to wound healing by utilizing proteases in the wound environment that are implicated in chronic wound pathophysiology. For example, chronic wounds (diabetic foot ulcers, pressure ulcers, venous stasis ulcers, and ischemic ulcers) are characterized by persistent inflammatory stimuli such as repeated trauma, ischemia, or low-grade bacterial contamination. In all these wounds, including pressure ulcers, the skin barrier is broken and bacterial colonization occurs and stimulates inflammatory cells such as neutrophils and macrophages to enter the wound. These activated inflammatory cells then secrete inflammatory cytokines such as TNF-α and IL-1 (which synergistically increase production of MMPs while reducing synthesis of TIMPs). The elevated protease secretion (MMPs, elastase, plasmin) degrades the ECM which interferes with cell migration and connective tissue deposition. Proteases also degrade growth factors and their target cell receptors which further limits the progression of the wound healing by eliminating the mediators of the cascade. Entry into the repair phase is thereby impaired, and the wound fails to heal. This differs from acute wounds in that there is a limited pro-inflammatory stimulus rather than ongoing stimulation as proposed in chronic wounds.

[0089] PEAU utilizes proteases in the wound environment to enzymatically degrade its constituent chains. PEAU has been shown to degrade following the action of proteases such as MMPs and elastases. Decreasing the amount of proteases in the environment is known to promote wound healing. FIG. 1 illustrates the enzymatic biodegradation of PEAU microcapsules through scanning electron micrographs of PEAU microcapsules in the presence of PBS, elastase, lipase, α-chymotrypsine, pancreatin, proteinase K, horse blood, and sheep blood.

[0090] In addition, the electrical properties of the side chain residues of PEAU positively impact the interaction of phages and the supported cells and promote wound healing. Positively charged PEAs, especially cationic 1-Lys-derived PEAs, provide a basis for materials to obtain relatively better biological properties, by interacting electrostatically with negatively charged cell membranes, thus promoting cellular internalization, cell adhesion, survival, growth, proliferation, differentiation, and migration for trauma repair and function reconstruction of tissues/organs. An additional secondary benefit of using polyester amides is that the degradation products (zwitterionic amino acids and diols, and relatively weak fatty di-acids) activate production of host growth factors and thus accelerate wound healing.

[0091] An in vivo full thickness study was performed on rats, which showed more efficacious tissue reactions for BACTELIDE than control, mediated in part by increased macrophage activation and stimulation of endothelial cell and smooth muscle cell proliferation. This is followed by an increase in myofibroblast population and neovascularization. The tissue deposition results in a collagen formation with fibrous band filling the surgically created wound in the dermis, subcutis, and panniculus muscle, covered with intact epidermis and merging with the pre-existing dermal collagen to either side. Stimulation of the tissue response was visualized by microcapsules embedded in the deep extent of the scar tissue, parallel to the skin surface surrounded by multifocal to coalescing small dense aggregates of macrophages (mild) and multinucleated giant cells (mild to moderate). Of interest is the presence of hypertrophic epidermidis in the control groups and its absence from the BACTELIDE treated group. Moreover, the Average Tissue Response Score (ATRS) was notable different between test article (males: 3.33; females: 4.66) and control (males: 5.66; females: 4.66) for the Day 43 groups, observing a lower score for specimen subjected to test article. These observations are likely to be an indication of the role of PEAU in immune stimulation and wound healing.

[0092] It is hypothesized that a beneficial effect of polyester amides is that the degradation products (zwitterionic amino adds and diols, and relatively weak fatty diacids) activate production of host growth factors and thus accelerate wound healing. An in vivo full thickness study showed a more efficacious tissue reactions than control mediated in part by increased macrophage activation and stimulation of endothelial cell and smooth muscle cell proliferation. This is followed increase in myofibroblast population and neovascularization. The tissue deposition results in a collagen formation with fibrous band filling the surgically-created wound in the dermis, subcutis, and panniculus muscle, covered with intact epidermis and merging with the pre-existing dermal collagen to either side. Stimulation of the tissue response was visualized by microcapsules embedded in the deep extent of the scar tissue, parallel to the skin surface surrounded by multifocal to coalescing small dense aggregates of macrophages (mild) and multinucleated giant cells (mild to moderate).

[0093] Amino acid-based biodegradable polymer encapsulation of active ingredients provides site of action delivery for increased bioavailability, prolonged release, and better compliance. BACTELIDE is non-toxic, non-irritants, biocompatible, hemocompatible, non-genotoxic, and does not exhibit in vivo local or systemic toxicity. BACTELIDE has been shown to promote tissue response, increase healing, and penetrate in deeper layers of the dermis. BACTELIDE allows for both superficial epidermis prolonged release and penetration promotion in the dermis PEAU biodegrades evenly and is stable at room temperature. PEAU and the other polymers described herein can be formulated into sprays, patches, thin films, powders, and incorporated into hydrogels, creams or suspensions, which are believed to promote also wound healing as described in the present example.

[0094] PEAU has several advantages over non-degradable polymers as well as degradable polymers such as PLLA and PLGA. Amino acid-based biodegradable polymers (AABBPs) are entirely composed of non-toxic building blocks such as naturally occurring amino acids and fatty diols and dicarboxylic, acids. These compounds contain hydrolysable ester bonds at a monomer stage, which when incorporated into the polymeric backbones are responsible for the biodegradation of the polymers.

[0095] AABBPs have many advantages over biodegradable polyesters (PEs) (polyglycolic and polylactic acids)) and their copolymers, including:

Polycondensation synthesis without using any toxic catalyst;
Higher hydrophilicity and, hence, better compatibility with tissues;
Longer shelf-life;
A wide range of desirable mechanical properties—from viscose-flow to strong materials with modulus of elasticity up to 6.0 gPa;
A variable hydrophobicity/hydrophilicity balance suitable for constructing devices suitable for sustained/controlled drug release;
An erosive mechanism and in vitro biodegradation rates ranging from 10-3 to 10-1 mg/(cm2.Math.h) that can be regulated by the addition of enzymes;
The vast majority of AABBPS are amorphous and biodegrade completely and evenly;
Excellent adhesion to plastic, metal, and glass surfaces.

[0096] In addition, it has been shown that polyester degradation by-products are acidic resulting in undesirable side-effects at a cellular level, which limits their use as functional tissue engineering scaffolds. In contrast to polyesters, AABBPs degradation bi-products are less acidic and more biocompatible. For example, hydrolysis products of polyester amide)s are neutral (zwitterionic) amino acids, readily metabolizable diols, and weak fatty acids. Another example are the biodegradation products of poly(ester urethane)s and poly(ester urea)s which are naturally, occurring physiological compounds such as CO2, hydrophobic amino acids, and diols. Polyester amides are known to be biocompatible, hemocompatible, and are used in stent-based local drug delivery.

[0097] When used, microcapsules are particularly advantageous for treatment as they penetrate deeply in the dermis, enhancing the above-describe properties in the tissue, and are not present only at its surface. FIG. 3 clearly shows the round empty spaces left deep in the tissue by microcapsules in a histological H&E stain of a wound treated with BACTELIDE, which illustrates deep penetration. Such empty spaces are absent from controls. FIG. 4 is the same image processed by halftoning for better reproducibility in the issued patent.

Toxicity Studies

[0098] Genotoxicity and mutagenicity assays according to the ISO 10993-3:2014 standard were performed to evaluate the mutagenic and carcinogenic potential of the extraction product of the PEAU polymer and the extraction product of the microcapsules (to assess the mutagenic potential of leachable substances or residues). Appropriate sample preparation procedures were done according to ISO 10993-3:2014(E) decision tree. The mutagenicity potential of the different samples was evaluated using the Ames modified ISO assay and the Pour plate Ames assay (with and without metabolic activation).

[0099] The following table shows that polymer extracts, and microcapsules extracts has no mutagenic potential (when we choose a significance level of 0.01).

TABLE-US-00001 TABLE 1 Results of the Ames test of undiluted (CC) «free PL-03-BM», polymer extracts, and microcapsules extracts expressed as Negative (N) or Positive (P) mutagenic potential on the full battery of bacterial strains. Polymer Microcapsules extracted in Polymer extracted in Microcapsules Test saline extracted in saline extracted in condi- solution PEG400 solution PEG400 tion a −S9 b +S9 c −S9 b +S9 c −S9 b +S9 c −S9 b +S9 c TA97a N N N N N N N N TA98 N N N N N N N N TA100 N N N N N N N N TA1535 N N N N N N N N WP2 N N N N N N N N a the results for undiluted (CC) samples were the same as the diluted ones (D1 .fwdarw. D4) b without metabolic activation c with metabolic activation

[0100] ISO 10993-18:2020 and ISO/TS 21726:2019 leachable studies were performed for BACTELIDE. Solutions of BACTELIDE were collected for analysis via the spraying. The extracts were analyzed by gas chromatography-mass spectrometry (GC-MS) for volatile to semi-volatile compounds, by liquid chromatography-mass spectrometry (LC-MS) for semi-volatile to nonvolatile compounds, and by inductively coupled plasma mass spectrometry (ICP-MS) for elemental (metal and other) components. After analysis, results were reported based on the evaluation criteria listed in the results section below. GC-MS based on the drug product extraction chromatographic data showed that five reportable compounds were identified in the test article extracts. 2-(N,N-Diethylamino)ethyldecyl maleate (CAS #184874-09-7) was the most abundant. Regarding LC-MS results, thirty-nine reportable compounds were identified in the test article extracts in positive ion mode. Glycerol dipentadecanoate (CAS #121957-69-5) was the most abundant. Thirty-six reportable compounds were identified in the test article extracts in negative ion mode. 3-[4-[[4-(2-Oxiranylmethoxy)phenyl]methyl]phenoxy]-1,2-propanediol didodecanoate (CAS #not given) was the most abundant. ICP-MS based on the drug product extraction elemental qualitative scan led to the identification of fifteen elements in the test article extracts. The most abundant element was magnesium.

[0101] A toxicological evaluation to assess the safety of the results reported, as they relate to the duration of exposure and nature of contact of the device, was performed to complete analysis of risk. A toxicological evaluation of leachable chemicals was performed to support safety and biocompatibility of the BACTELIDE formulation during its intended use in accordance with ISO 10993-1:2018, Biological Evaluation of Medical Devices, Part I: Evaluation and Testing Within a Risk Management Process (15010993-1, 2018); and FDA Guidance, Use of International Standard ISO 10993-1, “Biological evaluation of medical devices—Part 1: Evaluation and testing within a risk management process” (FDA, 2020). The safety question addressed in this risk assessment is whether intermittent, long term patient exposure to the microcapsule spray and levels of leachables from the test article could produce unacceptable human health risks, including carcinogenic and systemic non-carcinogenic risks.

[0102] In order to assess potential health risks posed by the test article, worst-case exposure estimations, including daily exposure and 100% bioavailability, were used to assess potential health risk. Solutions of drug product as contained in the submitted vials were collected for analysis via the spray actuation of the container and analyzed by GC-MS for volatile to semi-volatile compounds, by LC-MS for semi-volatile to nonvolatile compounds, and by ICP-MS for elemental (metal and other) components. All identified chemicals were assessed for risk.

[0103] The MOS values for all chemicals (or groups of similar chemicals) and elements identified across the analytical methods ranged from 3 to 1340000. A few chemicals had calculated “near-1” (i.e. ≥1 but <10) MOS values. As a group, the 3-[4-[[4-(2-Oxiranylmethoxy)phenyl]methyl]phenoxy]-1,2-propanediols had the lowest MOS value of three (3). However, the POD was based on body weight loss and elevated serum cholesterol. The gross findings and serum chemistry were not associated with adverse histopathology. Coupled with the IARC class 3 designation, the severity is interpreted as low and an acceptable exposure risk. Two chemicals (1,5-Dihydro-4-hydroxy-5-(phenylmethylene)-2H-pyrrol-2-one related compound and Dihydroxyphenyl trihydroxy-mono-carboxyacetylhexoside benzopyran-4-one) had MOS values of 5. Their margins of safety were calculated using the ICH M7 less than 1 year TTC of 0.33. Since exposure to product is intermittent through 56 days and because derivation of TTC levels is based on extrapolation from known compounds, the resulting MOS values are inherently conservative, which potentially overestimates the level of risk identified from the MOS calculations. Overall, there were no chemicals identified by GC-MS, LC-MS, or ICP-MS at levels of toxicological concern.

[0104] The risk of adverse health effects resulting from exposure to a material/product is dependent, among other factors, on the toxicological profiles of the chemical constituents and the nature and duration of contact. This study demonstrates acceptable worst-case margins of safety in the adult patient population for all identified chemicals, chemical groups, and elements from the PEAU microcapsule spray.

Nanocapsules

[0105] PEAU and the other polymers described hereinabove can be provided in the form of nanocapsules to treat the wounds, instead of the microcapsules. Nanocapsules have be prepared as follows. 30 mL of PVA 10% was poured in a 100 mL beaker. The first emulsion was prepared by adding 1.2 mL of Kolliphor 1188 1% in 12 mL PEAU in DCM (12.5% (w/v)) and setting the homogenizer at 35,000 rpm for 15 min on ice. The second emulsion was prepared by adding the first emulsion in the beaker containing the PVA and homogenizing as described above. Finally, DCM was evaporated through overnight magnetic bar stirring. The particle size distribution is illustrated in 5. D10, D50 and D90 particle size distributions were respectively 0.4053, 0.5759 and 0.9283 nm. Therefore, a particle size distribution with a D90 of less than 1 nm was obtained. Nanoparticles are smaller and therefore have more permeation in the wound tissues than microparticles.

[0106] Although the present invention has been described hereinabove by way of exemplary embodiments thereof, it will be readily appreciated that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. Accordingly, the scope of the claims should not be limited by the exemplary embodiments, but should be given the broadest interpretation consistent with the description as a whole. The present invention can also be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.