Topical Drug Delivery in the Treatment of Skin Wounds and Ocular Trauma Using the Platform Wound Device.

Topical treatment of injuries such as skin wounds and ocular trauma is the favored route of administration. Local drug delivery systems can be applied directly to the injured area, and their properties for releasing therapeutics can be tailored. Topical treatment also reduces the risk of adverse systemic effects while providing very high therapeutic concentrations at the target site. This review article highlights the Platform Wound Device (PWD) (Applied Tissue Technologies LLC, Hingham, MA, USA) for topical drug delivery in the treatment of skin wounds and eye injuries. The PWD is a unique, single-component, impermeable, polyurethane dressing that can be applied immediately after injury to provide a protective dressing and a tool for precise topical delivery of drugs such as analgesics and antibiotics. The use of the PWD as a topical drug delivery platform has been extensively validated in the treatment of skin and eye injuries. The purpose of this article is to summarize the findings from these preclinical and clinical studies.

Human platelet lysate delivered via an ocular wound chamber for the treatment of corneal epithelial injuries.

Current strategies to address corneal surface defects are insufficient to successfully resolve damage caused by injury and/or disease. To address this issue, we have developed an ocular wound chamber (OWC) that creates a fluid-filled environment by encompassing damaged ocular and periocular tissues allowing for the continuous delivery of therapeutics. This study tested human platelet lysate (hPL) as a treatment for corneal epithelial defects when used with the OWC. Corneal epithelial injuries were created in anesthetized guinea pigs by debridement of the central cornea. An OWC was placed over the injured eye and animals randomly grouped followed by injection of either 20% hPL, 100% hPL, or vehicle (balanced salt solution, BSS) into the chamber. Eyes were assessed at 0, 24, 48, and 72 h using intraocular pressure (IOP), optical coherence tomography (OCT), and fluorescein imaging. Whole globes were histologically processed, and hematoxylin and eosin (H&E) stained. No differences in IOP were recorded as a result of corneal wounding, chamber placement, and/or therapeutic application. OCT images demonstrated increased corneal swelling at 48 h and 72 h in the vehicle group compared to 20% hPL. Fluorescein staining showed increased corneal re-epithelialization in the 20% and 100% hPL groups at 48 h compared to vehicle only. H&E staining revealed increased stromal cellular infiltrate in the BSS group. This study demonstrates the delivery of hPL via the OWC improves corneal re-epithelialization and supports the expanded usage of the chamber in combination with hPL to manage a variety of corneal surface injuries, diseases and/or periocular conditions.

Treatment of Corneal Infections Utilizing an Ocular Wound Chamber.

Purpose: To demonstrate that the ocular wound chamber (OWC) can be used for the treatment of bacterial keratitis (BK). Methods: A blepharotomy was performed on anesthetized, hairless guinea pigs to induce exposure keratopathy 72 hours before corneal wound creation and Pseudomonas aeruginosa inoculation. Twenty-four hours postinoculation, eyes were treated with an OWC filled with 500 µL 0.5% moxifloxacin hydrochloride ophthalmic solution (OWC), 10 µL 0.5% moxifloxacin hydrochloride drops (DROPS) four times daily, or not treated (NT). White light, fluorescein, and spectral domain optical coherence tomography (SD-OCT) images; ocular and periocular tissues samples for colony-forming units (CFU) quantification; and plasma samples were collected at 24 and 72 hours posttreatment. Results: White light, fluorescein, and SD-OCT imaging suggests OWC-treated eyes are qualitatively healthier than those in DROPS or NT groups. At 24 hours, the median number of CFUs (interquartile range) measured was 0 (0-8750), 150,000 (106,750-181,250), and 8750 (2525-16,000) CFU/mL for OWC, NT, and DROPS, respectively. While 100% of NT and DROPS animals remained infected at 24 hours, only 25% of OWC-treated animals showed infection. Skin samples at 24 hours showed infection percentages of 50%, 75%, and 0% in DROPS, NT, and OWC groups, respectively. OWC-treated animals had higher moxifloxacin plasma concentrations at 24 and 72 hours than those treated with drops. Conclusions: OWC use resulted in a more rapid decrease of CFUs when compared to DROPS or NT groups and was associated with qualitatively healthier ocular and periocular tissue. Translational Relevance: The OWC could be used clinically to continuously and rapidly deliver antimicrobials to infected ocular and periocular tissues, effectively lowering bacterial bioburdens and mitigating long-term complications.

Use of an ocular wound chamber for the prevention of exposure keratopathy in a guinea pig model.

Current therapies available to treat and heal ocular surface injuries and periocular burns are frequently inadequate, costly, and labor intensive. To address these limitations, we have employed a flexible, semitransparent ocular wound chamber (OWC) to provide protection as well as a watertight seal to allow for the constant delivery of therapeutics to the ocular surface and surrounding periocular tissue. This study demonstrates the safety and utilization of the OWC on uninjured eyes and in our exposure keratopathy model. For initial safety studies (N = 3 per group), the eyelids remained intact and the eye uninjured. A blepharotomy (N = 6 per group) was performed to remove the upper and lower eyelids surrounding the left (OS) eye to create our exposure keratopathy model. Right (OD) eyes served as uninjured controls in all studies. Following OWC placement, 0.5 mL HPMC gel or balanced saline solution (BSS) was injected into the chamber. Animals were monitored daily and fully assessed via white light, fluorescein, and OCT imaging at least through 72 hours post OWC placement. In studies that included a blepharotomy, skin samples were analyzed by multiplex cytokine analysis. Results of safety experiments revealed no significant differences between treatment groups in corneal thickness, fluorescein staining, OCT imaging, or histological eye or skin sections when compared to control eyes. In our exposure keratopathy model, OWC treated eyes showed significantly less fluorescein uptake and also were found to have significantly lower levels of cytokines IL-13 and IL-5 in skin samples. These results demonstrate for the first time that treatment using the OWC device is not only safe, but significantly protects against blepharotomy-induced exposure keratopathy. As a whole, this study advances our overall efforts to develop a feasible solution to treat ocular surface injuries, infections, and periocular burns.

The utilization of an ocular wound chamber on corneal epithelial wounds.

PURPOSE: Currently available ocular moisture chambers are not adequate to manage the treatment of periocular burns, corneal injuries, and infection. The purpose of these studies was to demonstrate that a flexible, semi-transparent ocular wound chamber device adapted from technology currently used on dermal wounds is safe for use on corneal epithelial injuries. MATERIALS AND METHODS: A depilatory cream (Nair™, 30 seconds) was utilized to remove the excess hair surrounding the left eyes of anesthetized Institute Armand Frappier (IAF) hairless, female guinea pigs (Crl:HA-Hrhr). A 4 mm corneal epithelium defect was created using a corneal rust ring remover (Algerbrush®II). Epithelial defects were either left untreated or the eyes were fitted with an ocular wound chamber and 0.5 mL of hydroxypropyl methylcellulose (HPMC) gel (GenTeal®) or HPMC liquid (GenTeal®) was injected into each chamber (N=5 per group). At 0, 24, 48, and 72 hours fluorescein and optical coherence tomography imaging was collected and the intraocular pressure (IOP) was measured. H&E staining was performed on corneal and eyelid skin samples and evaluated by a veterinary pathologist. RESULTS: Corneal epithelial wounds demonstrated 100% closure rates when left untreated or treated with an ocular wound chamber containing HPMC gel at 72 hours while wounds treated with an ocular wound chamber containing HPMC liquid were 98% healed. No significant differences were found in corneal thickness and wound healing, IOP, or eyelid skin pathology in any treatment group when compared to controls. CONCLUSIONS: This study indicates that adapted wound chamber technology can be safely used on sterile, corneal epithelial wounds without adverse effects on periocular or ocular tissue when filled with a liquid or gel.

Treatment of corneal chemical alkali burns with a crosslinked thiolated hyaluronic acid film.

PURPOSE: The study objective was to test the utilization of a crosslinked, thiolated hyaluronic acid (CMHA-S) film for treating corneal chemical burns. METHODS: Burns 5.5mm in diameter were created on 10 anesthetized, male New Zealand white rabbits by placing a 1N NaOH soaked circular filter paper onto the cornea for 30s. Wounds were immediately rinsed with balanced salt solution (BSS). CMHA-S films were placed in the left inferior fornix of five injured and five uninjured animals. Five animals received no treatment. At 0h, 48h, 96h, and on day 14 post chemical burn creation, eyes were evaluated by white light imaging, fluorescein staining, and optical coherence tomography (OCT). Corneal histology was performed using H&E and Masson's Trichrome stains. RESULTS: Image analysis indicated biocompatible CMHA-S treatment resulted in significant decreases in the areas of corneal opacity at 48h, 96h, and on day 14 postoperatively. A significant increase in re-epithelialization was seen 14days post injury. CMHA-S treated corneas showed significantly less edema than untreated burns. No pathological differences were observed in corneal histological samples as a result of CMHA-S treatment. CONCLUSIONS: CMHA-S films facilitate re-epithelialization and decrease the area of corneal opacity in our corneal alkali burn rabbit model.

Bioactive Antimicrobial Peptides as Therapeutics for Corneal Wounds and Infections.

Significance: More than 2 million eye injuries and infections occur each year in the United States that leave civilians and military members with reduced or complete vision loss due to the lack of effective therapeutics. Severe ocular injuries and infections occur in varied settings including the home, workplace, and battlefields. In this review, we discuss the potential of developing antimicrobial peptides (AMPs) as therapeutics for the treatment of corneal wounds and infections for which the current treatment options are inadequate. Recent Advances: Standard-of-care employs the use of fluorescein dye for the diagnosis of ocular defects and is followed by the use of antibiotics and/or steroids to treat the infection and reduce inflammation. Recent advances for treating corneal wounds include the development of amniotic membrane therapies, wound chambers, and drug-loaded hydrogels. In this review, we will discuss an innovative approach using AMPs with the dual effect of promoting corneal wound healing and clearing infections. Critical Issues: An important aspect of treating ocular injuries is that treatments need to be effective and administered expeditiously. This is especially important for injuries that occur during combat and in individuals who demonstrate delayed wound healing. To overcome gaps in current treatment modalities, bioactive peptides based on naturally occurring cationic antimicrobial proteins are being investigated as new therapeutics. Future Directions: The development of new therapeutics that can treat ocular infections and promote corneal wound healing, including the healing of persistent corneal epithelial defects, would be of great clinical benefit.

Effect of Cationic Antimicrobial Protein CAP37 on Cytokine Profile during Corneal Wound Healing.

The cationic antimicrobial protein of 37 kDa (CAP37) mediates proliferation, migration, and adhesion of human corneal epithelial cells and promotes corneal re-epithelialization in mouse. The purpose of this study was to investigate the cytokine profile following abrasion of the corneal epithelium, and to identify the cytokines modulated by topical treatment with CAP37 to determine the mechanism by which CAP37 contributes to the recruitment of inflammatory cells and healing of the cornea. The corneal epithelium in mouse eyes was removed and wounds were treated with a saline vehicle or human recombinant CAP37. Wounds were visualized with fluoresce in staining at 0, 16, 24 and 48 h. Mouse corneas were excised at 0, 6, 16, 24 and 48 h post corneal abrasion. The excised corneas were analyzed by immunohistochemistry for re-epithelialization and infiltration of inflammatory cells while the expression profiles of thirty-two cytokines were investigated by multiplex analysis. Results corroborating previous studies showed accelerated wound closure in corneas treated with CAP37 compared to those treated with the saline vehicle. Immunohistochemistry revealed less neutrophil infiltration in CAP37-treated corneas when compared to controls at 24 h. By 48 h post-wounding, histological analysis revealed more staining for neutrophils than the staining observed in the controls. Modulation of cytokine expression occurred for the majority of the cytokines tested at the time of corneal abrasion, during re-epithelialization, and/or by CAP37 treatment. Cytokines monocyte chemoattractant protein-1 (MCP-1) and regulated on activation, normal T cell expressed and secreted (RANTES) were induced during re-epithelialization, at the early 16 h time point. Interleukin 6 (IL-6), leukemia inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF), IL-12p70, macrophage inflammatory protein 1 beta (MIP-1ß), and interferon gamma-induced protein 10 (IP-10) were induced at 24 h and unchanged during CAP37 treatment. By contrast, IL-15, monokine induced by gamma interferon (MIG), keratinocyte-derived cytokine (KC), tumor necrosis factor alpha (TNF-α), MIP-1α, IL-1ß, and macrophage colony-stimulating factor (M-CSF) were modulated by CAP37 treatment. In general, CAP37 appeared to decrease pro-inflammatory cytokines at 24 h and increase them at 48 h when compared to the control group. These data demonstrate that CAP37 modulates the production of cytokines in the cornea and suggest that limiting the number of neutrophils recruited during the early inflammatory phase may support corneal re-epithelialization.

Caveolin-1 modulates intraocular pressure: implications for caveolae mechanoprotection in glaucoma.

Polymorphisms in the CAV1/2 genes that encode signature proteins of caveolae are associated with glaucoma, the second leading cause of blindness worldwide, and with its major risk factor, intraocular pressure (IOP). We hypothesized that caveolin-1 (Cav-1) participates in IOP maintenance via modulation of aqueous humor drainage from the eye. We localize caveolae proteins to human and murine conventional drainage tissues and show that caveolae respond to mechanical stimulation. We show that Cav-1-deficient (Cav-1-/-) mice display ocular hypertension explained by reduced pressure-dependent drainage of aqueous humor. Cav-1 deficiency results in loss of caveolae in the Schlemm's canal (SC) and trabecular meshwork. However, their absence did not appear to impact development nor adult form of the conventional outflow tissues according to rigorous quantitative ultrastructural analyses, but did affect cell and tissue behavior. Thus, when IOP is experimentally elevated, cells of the Cav-1-/- outflow tissues are more susceptible to plasma membrane rupture indicating that caveolae play a role in mechanoprotection. Additionally, aqueous drainage from Cav-1-/- eyes was more sensitive to nitric oxide (NO) synthase inhibition than controls, suggesting that excess NO partially compensates for outflow pathway dysfunction. These results provide a functional link between a glaucoma risk gene and glaucoma-relevant pathophysiology.

A multifunctional peptide based on the neutrophil immune defense molecule, CAP37, has antibacterial and wound-healing properties.

CAP37, a protein constitutively expressed in human neutrophils and induced in response to infection in corneal epithelial cells, plays a significant role in host defense against infection. Initially identified through its potent bactericidal activity for Gram-negative bacteria, it is now known that CAP37 regulates numerous host cell functions, including corneal epithelial cell chemotaxis. Our long-term goal is to delineate the domains of CAP37 that define these functions and synthesize bioactive peptides for therapeutic use. We report the novel finding of a multifunctional domain between aa 120 and 146. Peptide analogs 120-146 QR, 120-146 QH, 120-146 WR, and 120-146 WH were synthesized and screened for induction of corneal epithelial cell migration by use of the modified Boyden chamber assay, antibacterial activity, and LPS-binding activity. In vivo activity was demonstrated by use of mouse models of sterile and infected corneal wounds. The identity of the amino acid at position 132 (H vs. R) was important for cell migration and in vivo corneal wound healing. All analogs demonstrated antimicrobial activity. However, analogs containing a W at position 131 showed significantly greater antibacterial activity against the Gram-negative pathogen Pseudomonas aeruginosa. All analogs bound P. aeruginosa LPS. Topical administration of analog 120-146 WH, in addition to accelerating corneal wound healing, effectively cleared a corneal infection as a result of P. aeruginosa. In conclusion, we have identified a multifunctional bioactive peptide, based on CAP37, that induces cell migration, possesses antibacterial and LPS-binding activity, and is effective at healing infected and noninfected corneal wounds in vivo.

Corneal wound healing, a newly identified function of CAP37, is mediated by protein kinase C delta (PKCδ).

PURPOSE: The neutrophil-derived granular protein, CAP37, an innate immune system molecule, has antibiotic and immunomodulatory effects on host cells, including corneal epithelial cells. We previously showed that CAP37 modulates corneal epithelial cell migration, adhesion, and proliferation, and that protein kinase C delta (PKCδ) mediates CAP37-induced chemotaxis of these cells. The objective of this study was to investigate the hypothesis that CAP37 facilitates corneal wound healing through the PKC signaling pathway. METHODS: The standard "scratch" assay performed on monolayers of corneal epithelial cells was used to measure the in vitro effect of CAP37 on wound closure. In vivo wound healing in response to CAP37 was measured using a mouse corneal epithelium abrasion model. In vitro and in vivo wound closure were monitored over 48 hours. The PKCδ was visualized during wound closure in cell monolayers and corneal epithelium by immunohistochemistry. The importance of PKCδ in CAP37-induced corneal wound healing was assessed by siRNA. RESULTS: We found that CAP37 accelerated wound closure in vitro and in vivo. Maximal closure occurred with concentrations of CAP37 between 250 and 500 ng/mL. Topical applications on mouse cornea led to re-epithelialization of the cornea by 24 hours. Immunohistochemistry of in vitro and in vivo wounds revealed a local increase of PKCδ along the wound edge in CAP37-treated cell monolayers and corneas, compared to untreated controls. CAP37-induced corneal wound healing was significantly reduced in vivo upon treatment with PKCδ siRNA. CONCLUSIONS: These findings support the hypothesis that CAP37 facilitates corneal wound healing through the activation of PKCδ.

CAP37 activation of PKC promotes human corneal epithelial cell chemotaxis.

PURPOSE: The objective of this study was to elucidate the signaling pathway through which cationic antimicrobial protein of 37 kDa (CAP37) mediates human corneal epithelial cell (HCEC) chemotaxis. METHODS: Immortalized HCECs were treated with pertussis toxin (10 and 1000 ng/mL), protein kinase C (PKC) inhibitors (calphostin c, 50 nM and Ro-31-8220, 100 nM), phorbol esters (phorbol 12,13-dibutyrate, 200 nM and phorbol 12-myristate 13-acetate, 1 µM) known to deplete PKC isoforms, and siRNAs (400 nM) before a modified Boyden chamber assay was used to determine the effect of these inhibitors and siRNAs on CAP37-directed HCEC migration. PKCδ protein levels, PKCδ-Thr(505) phosphorylation, and PKCδ kinase activity was assessed in CAP37-treated HCECs using immunohistochemistry, Western blotting, and a kinase activity assay, respectively. RESULTS: Chemotaxis studies revealed that treatment with pertussis toxin, PKC inhibitors, phorbol esters, and siRNAs significantly inhibited CAP37-mediated chemotaxis compared with untreated controls. CAP37 treatment increased PKCδ protein levels and led to PKCδ phosphorylation on residue Thr(505). Direct activation of PKCδ by CAP37 was demonstrated using a kinase activity assay. CONCLUSIONS: These findings lead us to conclude that CAP37 is an important regulator of corneal epithelial cell migration and mediates its effects through PKCδ.