The Development Of Gelatin And Recombinant Collagen Hydrogels As Novel Drug Sustained-release Agents And Effects On Prevention And Control Of Parasitic Diseases And Vectors

Parasitic diseases such as Schistosomiasis japonica and Angiostrongylus cantonensis are a class of zoonotic diseases associated with significant morbidity and mortality.The most effective strategies to combat parasitic diseases is to block transmission by killing the vectors that harbor infectious parasites,such as Oncomelania hupensis and Pomacea canaliculate for the above examples,respectively,and preventing parasitic infection using early drug treatment.The World Health Organization recommends using the molluscicide,niclosamide,and the antihelminthic drug,praziquantel,for the prevention and control of schistosomiasis in China.However,niclosamide leads to death of other aquatic organisms and praziquantel has a short half-life with low toxicity in humans.Thus,although these agents are the standard for the prevention and control of schistomiasis,they have significant limitations for broad application,and there is an urgent need for new approaches to control parasitic diseases.To address these limitations,in this study,we selected natural protein hydrogel（gelatin）as sustained-release drug carrier and characterized its properties,including protein sequence,internal structure,cytotoxicity,suitability for sustained-release of drug molecules,as well as its impact on the transmission medium.We also validated that gelatin hydrogels could be used to prevent parasitic infection by demonstrating that they could prevent cercaria infection in animal experiments and developed a gelatin-based drug sustained-release agent.On the other hand,We simultaneously conducted rational design of a recombinant collagen based on the triple-helical region of collagen Scl2 from Streptococcus pyogenes.The collagen domain（VCL）of Scl2 contains a spherical domain that was used as a functional module,and,to promote self-assembly and form a stable gelatin network,cysteines were introduced onto the N-terminal and C-terminal ends,and then additional cysteine residues were inserted into the collagen Scl2 domain.These adjustments in the amount of cysteine and the ratio of redox agents in the sequence yielded recombinant collagen hydrogels with controllable microstructure and mechanical properties.Finally,we developed an injectable,recombinant collagen hydrogel for sustained-release of drugs that effectively prevented anti-cercariae infection and was also effective for early treatment.The main research conclusions are shown as follows:（1）Prepared gelatin was characterized to determine its mechanical and temperature-controllable properties as well as to determine its behavior for sustained release of drugs.First,different concentrations of gelatin were prepared at concentrations of 1.0%(wv^-1)or above.Second,Circular Dichroism spectrum experiments demonstrated that the gelatin could fold correctly into the triplex structure.The melting temperature was approximately 38°C.The morphology of the self-assembled gelatin network was observed by Scanning Electron Microscope（SEM）,which showed that higher gelatin concentration resulted in smaller pores.Cell proliferation assays confirmed that gelatin could support growth of MC3T3-E1 cells and was not cytotoxic.In addition,a drug release experiment demonstrated that the concentration of gelatin influenced drug release;specifically,gelatin concentration less than 1.5%(wv^-1)did not readily absorb water and swell in solution,and this resulted in a more stable gel that could slowly release drug for up to 20 days.（2）The effects of sustained drug release from gelatin on parasite vector control and parasitic infection were evaluated.Different drug concentrations were coated with gelatin to determine the gel-forming and sustained-release properties corresponding to different concentrations of niclosamide and praziquantel.The gelatin-based sustained drug release capacity and efficacy of drug delivered by the hydrogels was confirmed.Microscopic analysis of 1 mg L^-1 niclosamide in sustained-release gelatin-based hydrogel in vitro demonstrated that it exerted a strong killing effect on snails within 48 hours and also completely killed cercariae within 30 minutes.This treatment also showed strong anti-cercariae infection activity in vitro:after 1 day of treatment,worms were reduced by about 80%and eggs were reduced by more than 60%.Notably,niclosamide-containing gelatin-based hydrogel can also be used as an effective supplement for anti-cercaria cream.In addition,1 mg L^-1 drug niclosamide in sustained-release gelatin-based hydrogel killed 100%of Pomacea canaliculate after 48 hours and reduced the hatching rate of eggs to only 28.5%of untreated groups.Finally,we assessed toxicity in a zebrafish experiment.The concentration of niclosamide in the aquatic environment did not increase during the sustained release period,and we did not observe any signs of toxicity in exposed zebrafish.（3）The recombinant collagen engineered with a triple-helix structure was obtained using rational design and construction approaches in addition to optimizing fermentation,expression,and purification parameters.Using the Scl2 collagen sequence VCL as a control,first,cysteine-containing disulfide bonds with redox-stimulation response function were introduced into the N-terminus and C-terminus of VCL.To promote cell adhesion,GFPGER was also introduced into the collagen domain,which resulted in an optimized S-VCL-S recombinant collagen protein.Subsequently cysteine was introduced into the middle,one-third,or two-thirds of the distance along the collagen region of Scl2 to yield recombinant collagen proteins S-VCL-S1,S-VCL-S2,and S-VCL-S3,respectively.The five proteins,VCLT,S-VCL-S,S-VCL-S1,S-VCL-S2,and S-VCL-S3 were expressed heterologously in E.coli BL21（DE3）.Using our optimized fermentation conditions,a maximum collagen yield of150 mg L^-1 was achieved.Collagen correctly folded into a triple-helix structure was obtained by affinity purification.The number of cysteine residues did not affect the secondary structure or thermal stability of the proteins.T_m values were between 37°C and 39°C.（4）The effect of redox agents on the formation of recombinant collagen hydrogels and their mechanical strength,shape,cytotoxicity,and drug sustained-release performance were characterized.Using inversion experiments,we showed that cysteine is indispensable for the formation of hydrogel networks,and 4%(w v^-1)recombinant collagen can self-assemble under the stimulation of 0.1%(w v^-1)hydrogen peroxide oxidation.The number of cysteines had a significant effect on the internal structure of the hydrogel,and the mechanical properties of the hydrogel was enhanced by increasing the number of cysteines in the collagen domain.SEM analysis revealed that a higher number of cysteine residues in the recombinant collagen correlated with more dense pore structure such that:S-VCL-S3>S-VCL-S2>S-VCL-S1>S-VCL-S.The number of cysteine residues in the collagen domain also affects the internal microporous structure of the hydrogel as well as the ability of the hydrogel to self-assemble.Cell proliferation assays confirmed that four of the designed recombinant collagen hydrogels could support growth of MC3T3-E1 cells with normal adhesion and proliferation and no observed cytotoxicities.Also,drug release experiments demonstrated that S-VCL-S,S-VCL-S1,S-VCL-S2,and S-VCL-S3 were suitable for use as sustained-release drug delivery systems,among which S-VCL-S3 performed the best.（5）The sustained-release properties of the recombinant collagen hydrogels and their ability to prevent and treat parasitic diseases were assessed.The purified recombinant collagen hydrogels were coated with different concentrations of drugs,which demonstrated that the drug coating did not affect the ability of the recombinant collagen to form a gel.Drug sustained-release tests confirmed that the recombinant collagen hydrogel could effectively control the release of niclosamide and praziquantel to kill larvae in vitro.The S-VCL-S3hydrogel containing 0.05 mg L^-1 niclosamide effectively killed cercariae within 24h,and increasing the concentration to 1 mg L^-1 niclosamide killed all cercariae within 30 minutes.In mice,niclosamide-containing recombinant collagen hydrogel smeared on the abdomen significantly reduced the rate of cercaria infection.When hydrogel-based delivery of niclosamide was combined with anti-larvae cream,both worms and eggs were completely killed after 24 hours and no animals were infected.Subcutaneous injection of praziquantel-containing recombinant collagen hydrogel robustly reduced the number of worms and eggs 24 hours before infection and 28 days after infection and showed strong efficacy in protecting the host liver.The results showed that sustained release of drugs from recombinant collagen hydrogel can kill cercaria and its adults in vitro and in vivo,which could effectively prevent and treat schistosomiasis infection.