| Skin is direct exposure to the external environment and is extremely vulnerable.If the wound is not treated until the wound heals by itself,it is easy to cause harmful consequences such as infection or scar.The process of healing of wound mainly relies on the synthesis and secretion of collagen and matrix by fibroblasts on the wound surface,which form granulation tissue together with new capillaries to fill the wound.The deposition of collagen is one of the signs of wound healing.Therefore,it is one of the means to increase the production and deposition of collagen,thus promote wound healing.Recombinant human collagen Ⅲ(Rhc-Ⅲ)is obtained by optimizing and recombining the original gene sequence of human collagen Ⅲ by genetic engineering technology,which is highly consistent with the amino acid sequence of natural human collagen.On the one hand,Rhc-Ⅲ has abundant hydrophilic groups,which can increase the adhesion and proliferation of fibroblasts.Moreover,it can directly supplement type Ⅲ collagen on the wound surface and reduce the risk of scars.However,water-soluble Rhc-Ⅲ is difficult to penetrate the lipid structure between stratum corneum cells and has a short action time.Therefore,it is necessary to rely on the carrier to penetrate the stratum corneum and prolong its action time to achieve its therapeutic effect.Liposomes(Lips)are tiny vesicles formed by encapsulating drug in a bilayer of lipids.The phospholipid structure of Lips is remarkably similar to the skin lipids of human body,with high biocompatibility and the ability to promote the transdermal absorption of drugs.Lips act on the lipid structure between stratum corneum cells to promote the penetration of drug through the stratum corneum,and can form a drug reservoir in the dermis to release drug slowly.In practical applications,the combination of Lips and gels can achieve the dual purpose of promoting drug penetration and skin adhesion.In this study,the type of the gel matrix is selected and the selected gel matrix is used to prepare the gel(Rhc-Ⅲ gels)firstly;Secondly,recombinant human collagenⅢ liposomes(Rhc-Ⅲ-Lips)were prepared by film dispersion method with egg yolk lecithin and cholesterol as lipid membrane materials.Rhc-Ⅲ-Lips were combined with the gel matrix to prepare recombinant human collagen Ⅲ liposomes gels(Rhc-Ⅲ-Lips gels).Thirdly,the quality,in vitro drug release and transdermal permeability of Rhc-Ⅲ gels and Rhc-Ⅲ-Lips gels were evaluated.Finally,the safety of animal skin and the pharmacodynamic experiment of rat were used to verify the advantages of Rhc-Ⅲ-Lips gels for wound healing treatment and provide better ideas and theoretical basis for the clinic.1.Preparation and evaluation of Rhc-Ⅲ gelsThe BCA method was used to determine the content of collagen and the methodological investigation results showed that the method meet the determination requirements.The optimal formulation and technology were obtained by single factor investigation,with appearance,viscosity,pH and uniformity as indexes.Three batches of reproducibility experiments were conducted to investigate the feasibility and stability of the formulation and process.The results showed that the optimal formulation and process had good reproducibility.The prepared Rhc-Ⅲ gels were colorless and transparent semi-solid.The content was(1.51±0.06)mg/g,the dynamic viscosity was(135.28 ± 7.25)Pa·s and pH was(5.98±0.02).The preliminary stability test results showed that Rhc-Ⅲ gels can keep stable under high humidity,strong light and high-speed centrifugation,but they need to be stored in a cool place(≤20℃)2.Preparation and characterization of Rhc-Ⅲ-Lips gelsFirstly,the dialysis method was chosen to determine the EE,DL and in vitro release of Rhc-Ⅲ-Lips and the method was investigated.Then,Rhc-Ⅲ-Lips were prepared by film dispersion method and the optimal prescription and technology were screened through single factor experiments with size,EE and DL as indicators.Finally,Rhc-Ⅲ-Lips gels were prepared by mixing Rhc-Ⅲ-Lips and blank gel matrix.The results showed that the dialysis method can separate lips and free drug.Rhc-Ⅲ-Lips was milky white suspension and showed a spherical shape with uniform particles under TEM.Particle size was(261.2±12.4)nm,the zeta potential was(-8.28±0.25)mv,the EE was(84.18±2.46)%,the DL was(6.31 ±0.15)%and pH was(6.27±0.05).The in vitro release results showed that the release of Rhc-Ⅲ sol cumulative release percentage reached(77.68±1.03)%,while the release of Rhc-Ⅲ-Lips was relatively slow with only(65.86±6.38)%released in 24 h and after 24 h,the drug was still being released slowly.Rhc-Ⅲ sol and Rhc-Ⅲ-Lips in vitro release process accorded with Weibull formula.The results showed that Rhc-Ⅲ-Lips gels was milky white semi-solid,the dynamic viscosity was(124.70±2.61)Pa·s and pH was(6.27±0.02).The preliminary stability test results showed that Rhc-Ⅲ-Lips gels can keep stable under high humidity,strong light and high-speed centrifugation,but they need to be stored in a cool place(≤20℃).3.In vitro release and percutaneous penetration test of Rhc-Ⅲ-Lips gelsBy using diffusion pool with pH 7.4 PBS as release medium,in vitro release study of Rhc-Ⅲ gels and Rhc-Ⅲ-Lips gels were conducted.The samples were taken out at a predetermined time and the content of collagen was determined.The cumulative release percentage was calculated and the Q-t curve was drawn.Then,the release of preparation in vitro was fitted by mathematical model.The in vitro release results showed that the cumulative release percentages of Rhc-Ⅲ gels and Rhc-Ⅲ-Lips gels for 24 h reached(73.47±1.74)%and(60.80±1.69)%respectively.The cumulative release percentage of Rhc-Ⅲ-Lips gels for 48 h reached(71.40 ±1.61)%,indicating that Rhc-Ⅲ-Lips has a certain sustained release effect.The in vitro release process conformed to Weibull formula,and the fitting coefficients were 0.9550 and 0.9812,respectively.By using diffusion pool with pH 7.4 PBS as percutaneous receptor fluid,in vitro percutaneous penetration study of Rhc-Ⅲ gels and Rhc-Ⅲ-Lips gels was conducted.The cumulative amount of transdermal was calculated and a transdermal curve was drawn to explore its in vitro transdermal penetration characteristics.The results showed that in intact skin,the cumulative transdermal amount of Rhc-Ⅲ gels and Rhc-Ⅲ-Lips gels for 24 h was(103.70±15.40)μg/cm2 and(134.70±8.21)μg/cm2 respectively.The cumulative transdermal amount of Rhc-Ⅲ-Lips gels was 1.30 times of Rhc-Ⅲ gels.In damaged skin,the cumulative transdermal amount of Rhc-Ⅲ gels and Rhc-Ⅲ-Lips gels in 24 h were(135.32±12.84)μg/cm2 and(156.21 ± 13.14)μg/cm2 respectively.The cumulative transdermal amount of Rhc-Ⅲ-Lips gels was 1.15 times of Rhc-Ⅲ gels.It showed that after Rhc-Ⅲ was made into liposomes,its transdermal amount can be increased.4.Skin safety and pharmacodynamics of Rhc-Ⅲ-Lips gels in animalsTo investigate the skin safety of Rhc-Ⅲ-Lips gels through the skin irritation test of rabbits and the skin allergy test of guinea pigs.The results showed that Rhc-Ⅲ-Lips gels have no skin irritation and allergies.This preparation is safe and can be used on damaged skin.Firstly,two round full-thickness skin defect wound surfaces with a diameter of 1.0 cm were made at the bilateral symmetry of the dorsal spine of rats to prepare the acute wound model.Then,the rats were divided into five groups(A:normal control group,B:blank gels,C:blank Lips gels,D:Rhc-Ⅲ gels,E:Rhc-Ⅲ-Lips gels).They were applied to the left wound,and the right wound was treated as a model control group without any treatment.Finally,observing the appearance of the wounds,taking photos and the healing rate of the wounds were calculated.Rats were sacrificed at preset time points and the skin at the wound site was stained by HE,MASSON and Sirius red respectively.The results of pharmacodynamics in rats showed that the shape,size and depth of the wounds in each group were uniform after modeled,indicating that the modeling method was reliable.The body weight of rats in each group increased at the end of the experiment,which can indicate to a certain extent that the preparation had no acute toxicity.The wound healing rate of Rhc-Ⅲ-Lips gels group was higher than that of other experimental groups on 3,7 and 12 days.The results of HE staining showed that compared with the model control group,the granulation tissues of Rhc-Ⅲ-Lips gels group appeared earlier and matured quickly,the epidermis could be completely epidermal in 12 days.The results of MASSON staining showed that the amount of collagen fibers deposited in the Rhc-Ⅲ-Lips gels group was large and the wounds healed well.Sirius red staining results showed that the amount of collagen III in the Rhc-Ⅲ-Lips gels group was more,which could reduce the risk of pathological scars.In summary,Rhc-Ⅲ-Lips gels have been successfully prepared.Compared with Rhc-Ⅲ gels,the in vitro release of Rhc-Ⅲ-Lips gels had a significant sustained release effect and the cumulative transdermal amount was significantly increased;Rhc-Ⅲ-Lips gels had no skin irritation and allergies;When Rhc-Ⅲ-Lips gels were applied to the wound surface,the healing rate and amount of collagen fiber deposition increased and the content of type Ⅲ collagen was high.When the wound was completely healed,the ratio of type Ⅰ/Ⅲ collagen fibers was closer to that of normal skin,indicating that the application of Rhc-Ⅲ-Lips gels can reduce the risk of pathological scars. |
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