¹Shriners Hospital for Children, University of Texas Medical Branch, Galveston, Texas, USA

²Shriners Hospital for Children Department of Surgery, University of Texas Medical Branch, Galveston, Texas, USA

³Shriners Hospital for Children Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, USA

⁴Department of Neurosurgery, University of Texas Medical School, Houston, Texas, USA

Correspondence to: MG Jeschke, Shriners Hospital for Children, 815 Market Street, Galveston, Texas 77550, USA

Exogenous insulin-like growth factor-I (IGF-I) is known to improve the pathophysiology of a thermal injury, however, deleterious side-effects have limited its utility. Cholesterol-containing cationic liposomes that encapsulate complementary DNA (cDNA) are nonviral carriers used for in vivo gene transfection. We propose that liposome IGF-I gene transfer will accelerate wound healing in burned rats and attenuate deleterious side-effects associated with high levels of IGF-I. To test this hypothesis IGF-I gene constructs, encapsulated in liposomes, were studied for their efficacy in modulating the thermal injury response. Thirty adult male Sprague-Dawley rats were given a 60% TBSA scald burn and randomly divided into three groups to receive weekly subcutaneous injections of liposomes plus the lacZ gene coding for -galactosidase, liposomes plus cDNA for IGF-I and -galactosidase or liposomes plus the rhIGF-I protein. Body weights and wound healing were measured. Muscle and liver dry/wet weights and IGF-I concentrations in serum, skin and liver were measured by radioimmunoassay. Transfection was confirmed by histochemical staining for -galactosidase. Rats receiving the IGF-I cDNA constructs exhibited the most rapid wound re-epithelialization and greatest increase in body weight and gastrocnemius muscle protein content (P < 0.05). local igf-i protein concentrations in the skin were higher when compared to liposomes containing only the lacz gene (p < 0.05) transfection was apparent in the cytoplasm of myofibroblasts, endothelial cells and macrophages of the granulation tissue. liposomes containing the igf-i gene constructs proved effective in preventing muscle protein wasting and preserving total body weight after a severe thermal injury.

growth factor; insulin-like growth factor-I; liposomes; trauma; wound healing

Introduction

Gene transfection is a promising approach to the treatment of various clinical disorders. There are, however, several obstacles to overcome before this approach can be effective.¹ One major obstacle is the selection of an appropriate vector for gene delivery.² Viruses have been used as delivery vectors due to the specificity with which they can bind to and infect cells.² The most common viruses used for transfection have been the retroviruses, adenoviruses and adeno-associated viruses.^1,2,3 Viral infection-associated toxicity, immunologic compromise, and possible mutagenic or carcinogenic effects, however, make this approach potentially dangerous.¹ The use of naked DNA not encapsulated or plasmid DNA constructs alone, without viral genes present, have been used topically or delivered with a pneumatic 'gene gun'. Both have proven to be inefficient at transferring genes, perhaps due to the fragility of the naked DNA constructs in the extracellular environment and the traumatic consequences of 'gene gun' discharges on cellular integrity.^1,4

When liposomes were discovered by Bangham,⁵ their in vivo utility was largely met with skepticism.⁶ Although the nonviral nature of liposomes and their stability in vivo are attractive, their use has been limited to in vitro due to the low in vivo transfection efficiencies.⁴ Modification of the standard liposomal structure to a cationic structure and the inclusion of cholesterol, together with the use of cytomegalovirus promoters in the cDNA constructs used for gene transfer, have increased the efficacy and transgenic expression levels to those previously achieved with adenovirus constructs.^4,7

A thermal injury is a particularly severe form of trauma characterized by high cardiac output, increased oxygen consumption, and protein and fat wasting.⁸ This vulnerable hypermetabolic state compromises the immune system and attenuates wound healing.⁹ Insulin-like growth factor-I (IGF-I) is an anabolic growth factor shown to improve metabolic rate, gut mucosal function and protein loss after a burn injury.^9,10,11 IGF-I is known to mediate the actions of growth hormone in the hypermetabolic state by attenuating lean body mass loss, improving the immune response, attenuating the acute phase response, and by enhancing wound healing.^{9,12,13,14,15,16} A key determinant of patient outcome after a burn injury is the wound healing process.¹⁴ Fibroblasts and keratinocytes have IGF-I receptors which probably mediate IGF-I stimulation of mitogenicity and proliferative activity.^17,18 There are, however, side-effects, such as hypoglycemia, mental status changes, edema, fatigue and headache, that are caused by the large amounts of systemic IGF-I needed for the desired therapeutic effects. These adverse side-effects limit the therapeutic utility of IGF-I in the treatment of burns.^19,20

We propose that delivery systems that rely on gene constructs encapsulated in liposomes can be effective and safe alternatives for in vivo transient gene transfer. To test the efficacy of liposomal delivery of IGF-I gene constructs we measured their biological effects, in terms of body weight, wound healing rates, protein content in muscle and liver, and IGF-I protein concentrations in the skin, serum and liver of burned rats.

Results

Nine of 10 rats in each group survived the 60% TBSA scald burn and experimental drug injections with no evidence of deleterious side-effects. One in each group died or was dropped from the study for reasons unrelated to the experimental treatment. No signs of adverse side-effects could be found throughout the study period. No hypoglycemia or electrolyte imbalance was detected at death. Furthermore, there were no significant differences in the serum IGF-I and IGFBP-3 concentrations among treatment groups, and all were within levels observed in unburned rats. This indicated that either the amounts of IGF-I protein given were small enough that the serum IGF-I and IGFBP-3 levels were not affected, or that the IGF-I protein and the IGF-I cDNA constructs remained at or near the injection sites and did not diffuse to a significant extent. It should be mentioned that the sensitivity of the used RIA is 20 ng/ml, which may be too low to detect small differences between groups. There was no significant difference among groups for liver IGF-I protein levels, total liver weights, or liver dry/wet weight ratios.

Total body weight increased at nearly 1.5% per week for the first 4 weeks after burn in animals receiving the liposome-IGF-I cDNA construct (Figure 1), while those treated with the liposome-rhIGF-I protein showed a loss in body weight during the first 6 weeks. Rats receiving treatment of liposomes plus -galactosidase showed an increase in body weight of about 50% of that observed in the cDNA-treated rats. Nearly all burned rats lost weight 28 days after burn when the wound area eschar was removed (Figure 1). From 6 to 8 weeks after burn, all animals gained weight at approximately the same rate (2% of their total body weight per week).

After the eschar was removed, the area of burn wound re-epithelization could be identified. This area was significantly larger in rats receiving IGF-I cDNA when compared with the rhIGF-I protein treatment 4 to 8 weeks after burn or when compared with liposome treatment at 4, 7, and 8 weeks after burn (P < 0.05) (Figure 2). These results were confirmed by histological measurements of linear re-epithelization rates (Figure 3). Rats treated with IGF-I cDNA and rhIGF-I protein showed significantly more re-epithelization 4 weeks after the thermal injury when compared with liposomes containing the lacZ gene (P < 0.05). There were no differences between groups 8 weeks after burn.

Skin IGF-I concentrations 8 weeks after treatment were significantly different between groups and within groups for proximal versus distal skin biopsies (Figure 4). Proximal skin biopsies from rats treated with IGF-I cDNA or rhIGF-I protein showed higher IGF-I tissue concentrations (encapsulated IGF-I cDNA: 207.4 ± 13.7 ng/mg; encapsulated rhIGF-I: 195.3 ± 8.0 ng/mg) compared with treatment with liposomes plus -galactosidase (167.0 ± 2.1 ng/mg) (P < 0.05). In the cDNA and rhIGF-I protein groups, a significant difference between the proximal and distal biopsies was measured (Figure 4). For encapsulated cDNA, the proximal IGF-I protein concentration was 207.4 ± 13.7 ng/mg wet tissue versus the distal skin biopsy of 167.9 ± 4.8 ng/mg wet tissue and for encapsulated rhIGF-I protein treatment the mean proximal concentration of IGF-I protein was 195.3 ± 8.0 ng/mg wet tissue versus 157.7 ± 2.5 ng/mg wet tissue for the distal biopsy (P < 0.05). IGF-I levels at the injection site remain elevated 5 days after injection, suggesting that encapsulating rhIGF-I in liposomes protect IGF-I against degradation. Animals receiving liposomes plus the lacZ gene coding for -galactosidase showed no significant differences in the IGF-I protein concentrations between proximal versus distal skin biopsies (167.0 ± 2.1 ng/mg versus 163.1 ± 4.7 ng/mg, respectively). No differences could be shown between IGF-I protein concentrations measured in the distal skin biopsies from rats receiving IGF-I cDNA, rhIGF-I protein or liposomes (Figure 4).

Histologic sections of the wound showed highly cellular and vascular granulation tissue in the floor of the burn wound. Hair follicles and other epidermal appendages were absent in the center of the wound and degenerative changes were present near its edges. Focal mixed acute and chronic inflammatory reactions were also observed. The fine granular blue-green reaction product of the -galactosidase reaction was predominantly present in the granulation tissue. The reaction product, shown in Figure 5a, was predominantly cytoplasmic in and near the myofibroblasts, endothelial cells, and macrophages of the granulation tissue. Staining for -galactosidase protein was also present within macrophages in areas of inflammation. A small amount of -galactosidase reaction was found in the matrix of hair follicle in normal skin near the wound margin (Figure 5b). No reaction for -galactosidase could be shown in skin away from the wound margin (Figure 5c). The gastrocnemius dry/wet weight ratio was significantly greater in rats receiving IGF-I cDNA compared with those receiving rhIGF-I protein (Figure 6).

Discussion

Wound healing is of major importance to the survival and clinical outcome of burn patients.^14,21 There is evidence that rhGH improves wound healing through stimulation of IGF-I.^9,17,22,23 In turn, IGF-I enhances wound healing by stimulating collagen formation and mitogenicity in fibroblasts and keratinocytes by binding to their receptors.^17,23 In this study we show that the administration of liposome-encapsulated IGF-I cDNA constructs improved the area of burn wound re- epithelization as well as wound linear re-epithelization when compared with rhIGF-I protein or liposomes containing the lacZ gene for -galactosidase. We further demonstrated that animals treated with the IGF-I cDNA construct had higher IGF-I protein concentrations in proximal skin biopsies compared with animals receiving liposome plus lacZ gene treatment. The distal skin biopsies had significantly lower IGF-I protein concentrations for the IGF-I cDNA and IGF-I protein treatment groups. These observations establish that the transient IGF-I cDNA transfections resulted in the local expression of IGF-I protein within a small perimeter of the sites of injection. Given the cotransfection with the lacZ gene construct, a non-mammalian gene, and the co-expression of the -galactosidase protein in the same tissue sample, the source for the expressed IGF-I protein is most likely the localized transfection of the IGF-I cDNA construct in the skin. This is in agreement with reports of high levels of liposome concentration and liposome-mediated expression at sites of application.²⁴ We propose that the therapeutic benefits of transient liposomal IGF-I gene transfection as described in this article may be further enhanced by increasing liposomal doses and/or the number of injection sites around the wound edges, thus increasing the number of transfected cells and concomitant levels of gene expression and secretion within the traumatized area. As we did not observe any significant differences in serum IGF-I, IGFBP-3 or liver IGF-I concentrations, it is likely that the beneficial effects of the liposomal gene delivery treatment, in terms of preservation of body weight and reduced protein loss in the gastrocnemius muscle, are due to enhanced wound healing and improved cell recovery and not to systemic changes in circulating levels of IGF-I protein. The advantages of early wound closure, demonstrated in several clinical studies, include a diminished hypermetabolic burn response and a decrease in inflammatory mediators, such as IL-6, IL-8 and TNF.^25,26

Previous studies using liposomes to transport IGF-I protein and IGF-I cDNA, have demonstrated weight gains and increases in wound healing in burned rats.¹⁸ These studies, however, included recombinant human growth hormone (rhGH) which may directly or indirectly influence wound healing and body weight gains, thus making it difficult to ascertain if one or both of these growth factors were responsible for specific effects. In our blinded study, the use of IGF-I without rhGH gave similar weight and wound healing improvements lending validity to the use of IGF-I gene transfer as the effective growth factor.

All skin sections including the injection site of the liposomal IGF-I cDNA plus -galactosidase cDNA construct were processed for Bluo-Gal staining to confirm transfection. In this study uninjured skin served as a control. There was strict localization of -galactosidase activity to the granulation tissue underlying the wound sites, which was composed of spindle-shaped myofibroblasts, macrophages and growing small blood vessels. The cells, which stained consistently, were the myofibroblasts, the endothelial cells and the macrophages, including multinucleate giant cells. While the bulk of the staining for -galactosidase was cytoplasmic, some reaction product was observed outside cell boundaries. This could be due to enzyme release from dead cells or simple diffusion of the reaction product from the cellular environment. A small amount of reaction product was present in the matrix of hair follicle, which was consistent with the sites of injection.

Cholesterol-containing cationic liposomes do not display the cytotoxicity typically associated with the use of other cationic liposomes in vivo, and significantly increase infectivity and transgene expression to therapeutic levels.^4,7 IGF-I cDNA gene transfer via liposomal delivery protects the IGF-I cDNA construct from degradation and enhances IGF-I protein expression in a well-defined region. This delivery system seems optimal for the purpose of favorably modulating the burn-induced hypermetabolic response via transient increases in the local expression of IGF-I protein.⁷ We suggest that this causes a concurrent stimulation of IGFBP-3 protein synthesis and increased levels of the biological active complex IGF-I/IGFBP-3 locally without any concomitant supraphysiological increase in circulating levels of free IGF-I protein. The smaller amounts of IGF-I protein achieved by liposomal transfection are better suited to the paracrine modality that is required to achieve positive therapeutic outcomes without detectable adverse side-effects.

Although the reasons for the beneficial effects associated with the treatment with liposomes encapsulating the lacZ gene coding for -galactosidase are not fully understood, it may be due to an enhancement in the uptake of extracellular nutrients and the in situ encapsulation and protection of endogenous growth factors and cytokines elaborated locally as part of the hypermetabolic response. There are also direct beneficial effects of the liposomal lipid moieties on damaged cell membranes at the burn site. Thus, it is not known whether the observed effects of liposome-encapsulated rhIGF-I protein treatment are due to the liposomal component or reflect an IGF-I protein effect.

It should be noted that the first 28 days of treatment provide a critical period for therapeutic intervention. It would be of great interest to focus efforts on this shorter time period and to provide mechanistic explanations for the improved outcomes observed after liposomal delivery of IGF-I cDNA expression vectors. Optimization studies suitable to a more clinical setting are best carried out within the earlier time frame.

In conclusion, we have demonstrated that administration of cholesterol-containing cationic liposomes (DC-Chol) encapsulating an expression plasmid vector for IGF-I cDNA, given to rats with a 60% TBSA thermal injury was effective in enhancing wound healing, preserving lean body mass, increasing IGF-I skin protein concentrations, and decreasing muscle protein wasting. Thus, cholesterol-containing cationic lipoplexes are suitable and safe as a delivery system for transient IGF-I gene transfections. Because of the infrequent weekly injection and low doses required, adverse side-effects are markedly reduced compared with the currently used systemic treatment with recombinant protein products.

Materials and methods

Thirty adult male Sprague-Dawley rats (350-375 g) were placed in wire bottom cages and housed in a temperature-controlled room with a 12 h light-dark cycle. The animals were acclimatized to their environment for 7 days before the start of the blinded study. All received equal amounts of a liquid diet of Sustacal (Mead Johnson Nutritionals, Evansville, IN, USA) and water ad libitum throughout the study. This study was approved by the Animal Care and Use Committee of the University of Texas Medical Branch, Galveston, TX.

Each rat received a 60% total body surface area (TBSA) full-thickness scald burn as described by Herndon et al.²⁷ They were then randomly divided into three groups to receive:

1. weekly subcutaneous injections of liposomes (10 l liposomes in 180 l saline) plus 0.2 g of the lacZ cDNA construct coding for -galactosidase (n = 10);

2. weekly subcutaneous injections of liposomes (10 l liposomes in 180 l saline) containing 2.2 g of a cDNA coding for IGF-I and 0.2 g of a -galactosidase cDNA construct (n = 10);

3. weekly subcutaneous injections of liposomes (10 l liposomes in 180 l saline) containing 22.2 g rhIGF-I protein (n = 10).

Liposomes used were the cholesterol-containing cationic form (DC-chol).²⁸ The IGF-I cDNA construct consisted of a cytomegalovirus-driven IGF-I cDNA plasmid prepared at the UTMB Sealy Center for Molecular Science Recombinant DNA Core Facility by Dr T Wood. The IGF-I cDNA was a generous gift from Dr P Rotwein, NIH, Bethesda, MD, USA. Mixtures were prepared fresh every week before injections and assigned using a blind protocol. Codes were broken after all measurements were completed.

On the day of burn, each rat received 0.2 ml of one of the solutions injected at a site, just outside the burn wound, 1 cm from the wound margin. This was repeated once a week for 8 weeks. During the entire study period, animals were critically examined every day for adverse side-effects, such as behavior abnormalities, changes in hair quality, food intake or signs of infection. Body weights were measured at the same time each week. The wound eschar was left intact for the first 28 days and then removed by gentle traction, caution being taken not to disturb or destroy the healing edge along the periphery. Each week, after removing the eschar, the animals were placed on a standard surface and the wound area traced on to acetate sheets along the well-demarcated re-epithelized and non-burned interface and the leading edge of the neoepithelium. The areas of these tracings were calculated by computerized planimetry (Sigma Scan and Sigma Plot software, San Rafael, CA, USA). Skin biopsies were taken from the wound edge at 4 weeks and 8 weeks after burn and light microscopic analysis was performed using established techniques. Investigators were blinded to the agents given, during all measurements and data analysis.

Animals were killed by decapitation 5 days after the last injection (8 weeks after burn). Blood was collected into serum and plasma separators, and spun at 1000 g for 15 min, decanted and stored at -73°C. Liver, kidney, gastrocnemius muscle and dorsal skin samples were harvested, snap frozen in liquid nitrogen and stored at -73°C for analysis. The gastrocnemius muscle and liver were weighed and samples dried at 60°C to constant weight. The dry/wet weight ratios were used to estimate protein content. Histological measurements for linear skin re-epithelization used the HE staining technique. Serum glucose and electrolytes were determined using a Behring nephelometer (Behring, Dearfield, IL, USA). IGF-I and human IGFBP-3 protein serum concentrations were measured by RIA as described in the kit guidelines (Nichols Institute, Geneva, Switzerland). The IGF-I protein tissue concentrations were measured in liver and skin. IGF-I protein concentrations were measured in proximal (biopsy near the injection site) and distal (biopsy at the farthest point from the injection site) skin samples. Proteins were extracted by pulverizing approximately 40 mg tissue under liquid nitrogen, adding an extraction buffer (PBS^-, 0.25 ml PMSF, 50 mg leupeptin, 100 mg aprotinin and 50 mg antipain) in a volume 1:7 (7 ml buffer/g tissue) and homogenizing the mixture. After homogenization, 50 l of the homogenate was added to 150 l of extraction solution and centrifuged at 13 500 g for 5 min. 100 l of supernatant was added to 400 l of neutralization solution, and the IRMA assay performed as described in the kit guidelines (Diagnostic System Laboratories, Webster, TX, USA).

The presence of the -galactosidase protein was detected by histochemical staining with Bluo-gal for -galactosidase. Linear biopsies, approximately 2 mm thick, extended from the center of the burn wound well into surrounding normal skin. Skin specimens were fixed overnight at 4°C in fixative consisting of 4% paraformaldehyde in a Hepes-buffered Hank's solution at pH 7.6. After washing in buffer and phosphate-buffered saline (PBS), the specimens were incubated overnight at 37°C a 0.1% solution of Bluo-Gal substrate (halogenated indolyl--d-galactoside) (LIFE Technologies, Gaithersburg, MD, USA), buffered to pH 7.6. After extensive washing, tissues were embedded in paraffin, and histologic sections made and stained with hematoxylin and eosin or with eosin alone.

Statistical comparisons were made by ANOVA following Student's t test with Bonferroni's correction. Data are expressed as means ± s.e.m. Significance was acceptance at P < 0.05.

Acknowledgements

This study was supported by the Clayton Foundation for Research and the Shriners Hospital for Children. We would like to thank Anne S Burke, Robert A Cox, Drs Minas Chrysopoulo and Meelie DebRoy for their technical support.

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Figure 1 Per cent change in body weights are depicted for three therapies applied during the 8-week study. Liposome encapsulated IGF-I cDNA construct were significantly higher versus liposomes encapsulating the IGF-I protein from days 14 to 56, P < 0.05. Liposomes and liposomes encapsulating the IGF-I cDNA construct were different in the first week of therapy, P < 0.05. All animals lost weight 28 days after burn as a result of the removal of the burn eschar. Data are presented as means ± s.e.m. *cDNA versus IGF-I, ^†cDNA versus liposomes.

Figure 2 Area of wound re-epithelization measured by planimetry. Rats receiving encapsulated IGF-I cDNA constructs showed the highest per cent of re-epithelization throughout the study period compared with the liposomes or liposome encapsulated rhIGF-I protein. Data are presented as means ± s.e.m. *Liposome cDNA versus liposome IGF-I, P < 0.05. ^†Liposome cDNA versus liposomes, P < 0.05.

Figure 3 Linear wound re-epithelization determined by histology at 4 weeks after burn injury. *Liposomes versus IGF-I cDNA and rhIGF-I protein 4 weeks after burn injury, P < 0.05.

Figure 4 Mean IGF-I concentrations 8 weeks after receiving liposomes, liposomes plus rhIGF-I or liposomes plus the IGF-I cDNA in proximal biopsies (biopsy nearest injection site) and distal biopsies (biopsy farthest from the injection site). *Significant difference in IGF-I protein concentrations in the proximal biopsies of rats receiving encapsulated IGF-I cDNA constructs and encapsulated rhIGF-I protein compared with their distal skin biopsies and ^†to the proximal biopsies of rats receiving liposomes, P < 0.05.

Figure 5 Histologic sections of skin after histochemical reaction for demonstration of -galactosidase activity and counterstained with eosin. (a) Finely granular blue-green reaction product is present within many myofibroblastic and histiocytic cells in the granulation tissue underlying the burn wound. Original magnification ´380. (b) Uninjured skin near the edge of the burn wound shows small amounts of reaction product in the matrix of hair follicles. Original magnification ´380. (c) Control dermal tissue underlying uninjured skin distant from the burn wound contains no reaction product. Original magnification ´380.

Figure 6 Gastrocnemius muscle dry/wet weight ratios. *IGF-I cDNA constructs versus liposomes and encapsulated rhIGF-I, 8 weeks after receiving a scald burn injury, P < 0.05.