Synthesis And Investigation Of Multifunctional Recombinant Protein Drug Carrier For Treatment Of Skin Disease And Skin Lesion

Background:Skin cancer is a common disease of superficial tumors in plastic surgery.Its high incidence and mortality rate pose a serious threat to human health and even life.At present,the effect and prognosis of common clinical surgical treatment for aggressive skin cancers,for example,squamous cell carcinoma,are still unsatisfactory.Surgical resections for tumors with excessive volume,multiple metastatic or invade deep tissues will inevitably lead to deformed appearance and dysfunction.Traditional chemotherapy for systemic treatment,however,has more economic advantages than immunotherapy,but still have defects such as short drug half-life,low bio-availability,and severe drug toxicity to non-target organs.Recently,photothermal therapy has gradually become a research hotspot in tumor treatment due to its advantages of non-invasiveness and locality.A variety of near-infrared responsive inorganic nanomaterials was developed for tumor treatment by their efficient heating characteristics.But their clinical application was restricted by the local inflammation induced by ultra-high temperature,long-term risks that caused by the low biocompatibility and low metabolic degradation of inorganic materials.Therefore,it is of great significance to find new multifunctional,safe and effective therapeutic methods.Besides,skin scald is a common complication of non-invasive thermal ablation therapy and can be accompanied by skin infection in severe cases.Burns combined with infected wounds is also a difficult problem in the clinical treatment of plastic surgery.Although wound dressings can improve the effect of wound repair,they are failed to achieve the dual effects of antibacterial and drug loading to promote healing or even exert long-term effects.Thus the research on the development of multifunctional materials has important clinical value.Elastin-like protein is an artificial recombinant protein,which has unique polymer drug loading and elastic reversible phase transition properties.Its structural design is based on the elastin functional unit polypeptide,and has great functional potential and broad application prospects in long-acting drugs,nano-drug delivery system and gel sustained release.Further,Elastin-like protein has favorable biocompatibility and degradability.The fusion protein modified by genetic engineering has multifunctional biological integration which make it an excellent candidate for multifunctional biological drug delivery in many fields of medical diseases.The present research is designed and conducted based on the background above.Objectives:1.To construct and characterize recombinant protein bio-loaded near-infrared photoresponsive nanoparticles and study its photothermal effect,pharmacokinetic effect,toxic and side effects,and anti-skin cancer efficacy in vivo and in vitro.2.To construct and characterize recombinant protein bio-loaded hydrogel and study its biocompatibility,antibacterial effect,ability to promote cell proliferation and migration,and effect on promoting repair in a rat skin burn infection model.Methods:1.Designing and constructing recombinant protein K72 plasmid by genetic engineering method,transform and express in E.coli and obtain K72 protein through multi-step purification.The nano-protein drug is prepared by supramolecular self-assembly with carboxylated polyethylene glycol and cisplatin,and then self-polymerized with dopamine to form a polydopamine layer.The morphology and other physical properties of the nanoparticles were characterized by transmission electron microscopy,hydrated particle size,surface potential,and ultraviolet-visible light absorption measurements.Carrying out in vitro drug sustained-release experiments to detect drug sustained-release properties.And use 808 nm laser to irradiate the nanoparticle aqueous solution to monitor the heating effect and calculate the photothermal conversion efficiency.2.Carrying out cell proliferation experiments and live-dead cell staining experiments to evaluate the killing effect of nanoparticles on tumor cells in vitro,and calculate and compare the half inhibitory concentrations of various treatment methods.Conducting in vivo pharmacokinetic studies in rats to determine the half-life and bioavailability of nanoparticle drugs.The photothermal conversion effect was detected by subcutaneous injection of nanoparticles into mice and 808nm laser irradiation.Establishing a mouse skin squamous cell carcinoma model,monitor and record the mouse body weight and other body conditions and tumor growth during treatment.At the end of the observation period,blood samples were collected from the mice for blood biochemical testing to evaluate the liver and kidney function indicators in each group,and isolated tumors and organ tissues were collected for pathological analysis to evaluate the proliferation,apoptosis and organ tissues of tumor cells in each group.pathological changes.3.Designing and constructing recombinant fusion protein EGF-K72 plasmid by gene chimeric method,transform and express in Escherichia coli and obtain EGF-K72protein through multi-step purification.The intermediate protein EGFK72-SH was synthesized by modifying the protein through amide bond reaction,and finally it was gelled with silver nitrate and hyaluronic acid through Ag-S bond and electrostatic self-assembly to synthesize EGF-K72-Ag/HA hydrogel.The morphology,mechanical properties and other physical properties of the hydrogel were characterized by scanning electron microscope,infrared spectrum absorption,X-ray diffraction and rheological detection.In vitro sustained-release experiments were performed to detect the sustained-release performance of the hydrogel Ag⁺.4.Performing Cell proliferation experiments,cell migration experiments and live/dead cell staining experiments in vitro to evaluate the biocompatibility of the hydrogel and the effect on cell proliferation activity.Carrying out the Bacteriostasis tests by using methicillin-resistant Staphylococcus aureus and Escherichia coli,detecting the antibacterial ability of the hydrogel in vitro.Using the mouse skin irritation test to determine the Hydrogel biotoxicity.Establishing rats skin scald combined with infected wound model,monitor and record the weight change and wound healing of the rats during the treatment,and then calculate the wound healing rate.On the 7th,14th,and 21st days of the observation period,taking the blood biochemical testing to evaluate blood cells and related inflammatory indicators.At the end of the observation period,collecting wound tissue for pathological analysis to evaluate the outcome of inflammation and wound repair and remodeling.Results:1.K72-Pt-PEG-PDA（KPPD）nanoparticles with uniform morphology were synthesized by biological self-assembly strategy,with an average particle size of 109.6±20.9nm,a hydrated particle size of 459nm,and a surface potential of-3.6 m V.The spherical nanocarriers can effectively load cisplatin with a drug loading rate of 28.7%,and exhibit good sustained-release ability in vitro.The spectral absorption in the near-infrared region（700–900 nm）of KPPD nanoparticles showed high absorbance.After the KPPD aqueous solution was irradiated with an 808nm laser with a power of1.5W/cm2 for 10 minutes,the temperature rose to 74°C（ΔT=47.9°C）,and the photothermal conversion efficiency was 67.57%.2.The co-culture of nanocarriers with NIH/3T3 cells did not show cytotoxicity.When acting on SCC-7 cells,compared with the original drug cisplatin（Pt）group,pure photothermal（KPD）group,and nanoparticle（KPPD）group,the nanoparticle 808nm laser irradiation（KPPD+Laser）group showed the strongest The inhibitory effect on cell growth was consistent with the results of live and dead cell staining experiments.The IC₅₀values of Pt group and KPPD+Laser group were229.2μg/m L and 65.7μg/m L,respectively.The pharmacokinetic study results in rats showed that the effective half-life of KPPD increased to 54.2h,and the bioavailability increased to 241.6hμg/ml,which were 3.5 times and 2.3 times the corresponding indicators of the original drug,respectively.The results of in vivo experiments in squamous mice with skin confirmed that the KPPD+Laser group showed the best anti-tumor effect,and no tumor recurrence was observed.H&E staining and TUENEL staining of tumor tissues showed similar results,that is,the KPPD group showed the largest proportion of dead and apoptotic tumor cells.During the 16-day observation,the monitoring survival rate and body weight of the mice in the Pt group decreased to a certain extent,the liver and kidney function indicators in the blood biochemical index results were abnormally increased,and a certain degree of renal damage was observed in the renal histopathological sections,no obvious abnormalities were found in the KPPD group respect to the above results.3.EGF-K72-Ag/HA（EKA）hydrogel was synthesized by gene fusion and biological self-assembly strategy,which has shear thinning properties and viscoelasticity.The hydrogel showed a porous structure under the scanning electron microscope,and a characteristic absorption peak was detected at 1380cm^-1 in the infrared absorption spectrum.Under physiological conditions,the hydrogel exhibited Ag⁺sustained-release ability with degradation.The results of in vitro antibacterial experiments showed that EKA hydrogel inhibited the proliferation of Escherichia coli and methicillin-resistant Staphylococcus aureus in a time-and dose-dependent manner.4.When co-cultured with EKA hydrogel extract,the proliferation and migration.activities of NIH-3T3 cells were enhanced in a time-and dose-dependent manner.No biological toxicity was found in the results of mouse skin irritation test.The results of in vivo experiments on rats with skin scald infection confirmed that the wounds of the EKA treatment group showed the best repair effect.After 21 days of treatment,the wound healing rates of the control group,growth factor gel（EGF）group,protein gel（EK）group,and EKA hydrogel group were 77.6%,91.6%,90.0%,and 98.1%,respectively.On the 7th,14th,and 21st days of the observation period,the leukocytes and neutrophils counts in the EKA group were significantly lower than those in the other groups,and the rest of the blood cell counts were normal.Compared with the pathological results of wounds in each group at different times,the EKA group showed less inflammatory cell infiltration,earlier and faster epidermal re-epithelialization and dermal collagen regeneration.Besides,the results of immunohistochemistry showed that the expression of IL-6 in the EKA group was significantly lower than that of the other three groups,while the expression of TGF-βwas significantly higher than that of the other three groups.The results of immunostaining for pox light showed a consistent result,and col-1α,ELN,and CD31 in the EKA group showed the strongest pox light performance on average.Conclusions:1.A recombinant protein near-infrared responsive nanoparticle KPPD was prepared and synthesized,which has a strong anti-tumor effect of long-acting photothermal synergistic chemotherapy.In addition to high photothermal conversion efficiency and stability,the slow-release effect of the nanosystem improves the half-life and bio-availability of the drug,which can effectively reduce the frequency of drug administration for skin cancer treatment in vivo.The nanosystem utilizes degradable synthetic materials,exhibits excellent biocompatibility,and eliminates the toxic damage of traditional chemotherapy drugs to non-target organs effectively.2.The recombinant protein hydrogel EKA was prepared and synthesized,which can use the cohesive force and porous structure in the system to obtain the sustained release performance of the drug,and realize long-acting efficient wound repair for burn infection.The biodegradable biohydrogel,with multi-duty drug performance,has good biocompatibility and multifunctional effects of anti-inflammatory,antibacterial and wound remodeling.3.The present research proposes a novel assembly method for genetic bioengineering protein for drug-loaded,which achieves a simple and controllable preparation process by utilizing supramolecular forces.Synthetic protein biofunctional materials have the potential to be flexible and modifiable in structure and broad application prospects in the exploration of other related fields in the future.