A novel sustained-release agent based on disulfide-induced recombinant collagen hydrogels for the prevention and treatment of Schistosoma infections

Abstract

Schistosomiasis is commonly managed using the praziquantel, but it is only effective against adult worms and duration of action is short. Liver fibrosis will worsen if eggs are still present after stopping treatment. Therefore, this study aimed to develop a sustained drug release system for effectively preventing and treating schistosomiasis. A disulfide bond-induced three-dimensional (3D) recombinant collagen hydrogel was developed for sustained praziquantel release. Three collagen sequences were developed based on the sequence for Scl2 of S. pyogene, with different substitutions of residues for cysteine (S-VCL-S1, S-VCL-S2, and S-VCL-S3). Their properties were tested. Mice were infected with Schistosoma japonicum cercariae and treated either with praziquantel collagen hydrogel or niclosamide collagen hydrogel. The worm-killing effect was examined. The application of hydrogel-niclosamide on the skin for 24 h effectively prevented Schistosome cercariae from infecting mice and showed 70.95% and 81.73% reduction in the number of eggs and worms, respectively. The combined use of niclosamide and anti-cercariae cream showed 100% protection after 24 h. The hydrogel-praziquantel also showed a 100% reduction of worms and eggs after 24 h of subcutaneous injection. The subcutaneous injection of praziquantel after 28 days of infection showed 95.19% and 80.12% reduction of worm and egg counts, respectively, and the development of larvae was significantly slowed down. Liver analysis showed no infection after 7 days of treatment. These results suggest developing a novel type of sustained-release agent against schistosomiasis based on the recombinant collagen hydrogel that provides a potential new treatment for schistosomiasis.IMPORTANCEThis study introduces an new way for treating schistosomiasis: a special collagen hydrogel that gradually releases medication to treat schistosomiasis effectively. This innovation provides a promising way to treat schistosomiasis. It represents a significant step forward in the fight against this disease and offers hope for more effective and safer treatments in the future.

Keywords: praziquantel; recombinant collagen hydrogel; schistosomiasis; self-assembly; sustained drug release.

Fig 1

(A) Schematic illustration of the designed S-VCL-S1, S-VCL-S2, and S-VCL-S3 collagen protein with cysteine residues inserted at the N- and C- termini and the appropriate amino acids located in the middle and 1/3 and 2/3 of collagen are replaced by cysteine. (B) Molecular networks are created through the formation of the disulfide bonds between the cysteine residues. (C) The length of the nucleotide fragments after digesting the recombinant plasmids with BamHI and Nco I enzymes. M is the DNA marker. The left shows the intact pET-28a-S-VCL-S1-3 constructs, while the right shows the same constructs after double digestion with BamHI and Nco I. Molecular weights of proteins determined through SDS-PAGE analysis of the purified collagen proteins, S-VCL-S1-3. M shows standard protein marker; Left shows protein expression before purification of S-VCL-S1, SVCL-S2, and S-VCL-S3; Right shows S-VCL-S1, SVCL-S2, and S-VCL-S3 after purification. (C) MALDI-TOF mass spectrometry characterizing the molecular weights of S-VCL-S1, SVCL-S2, and S-VCL-S3 collagen.

Fig 2

CD characterizations of the designed proteins. (A) Far-UV CD spectra of the collagen proteins and (B) thermal denaturation profiles of S-VCL-S, S-VCL-S1, SVCL-S2, and S-VCL-S3 at 1 mg/mL in 10 mM phosphate buffer (pH 7.4).

Fig 3

(A) Storage and loss moduli, G′ and G″, of S-VCL-S, S-VCL-S1, S-VCL-S2, and S-VCL-S3 hydrogels at 4 wt.% in 10 mM PBS (pH 7.4). (Above) 2 wt.%; (below) 4 wt.%. (B) SEM micrographs of the collagen-like proteins before and after the addition of H₂O₂. (A–C) S-VCL-S, (D–F) S-VCL-S1, (G–I) S-VCL-S2, and (J–L) S-VCL-S3. Scale bar = 400 µm for A, B, D, E, G, H, J, and K and 100 µm for C, F, I, and L. The concentration of S-VCL-S, S-VCL-S1, S-VCL-S2, and VCL-S3 is 4 wt.% with PBS or without 0.1 wt.% H₂O₂. PS: (A–C) S-VCL-S in Figure B as control here are from our previously published article titled “Recombinant collagen hydrogels induced by disulfide bonds.”

Fig 4

Sustained release of (A) niclosamide and (B) praziquantel from the recombinant collagen hydrogel S-VCL-S3. (C) Fitting curve of S. cercariae mortality at different time points and different concentrations of sustained-release niclosamide recombinant collagen hydrogel. The difference was considered significant at P < 0.05 (n = 3).

Fig 5

(A) Egg and (B) worm reduction rates (%) of recombinant collagen hydrogel with sustained niclosamide release applied to the abdomen of mice before 4 to 72 h of S. cercariae infection. (C) Digital images of liver egg of mice coated with recombinant collagen hydrogel niclosamide sustained-release agent on abdomen before 4 to 72 h S. cercariae infection liver analysis.

Fig 6

(A) Egg and (B) worm reduction rates (%) in mice treated with subcutaneous injection of recombinant collagen hydrogel containing praziquantel sustained-release agent 4 to 24 h before S. cercariae infection. (C) Digital images of liver egg of mice coated with recombinant collagen hydrogel niclosamide sustained-release agent on abdomen before 4 to 72 h S. cercariae infection liver analysis.

Fig 7

(A) Egg and (B) worm reduction rates (%) in mice treated with subcutaneous injection of recombinant collagen hydrogel containing praziquantel sustained-release agent after 28 and 35 days of S. cercariae infection. (C) Digital images of the liver eggs of mice coated with recombinant collagen hydrogel praziquantel sustained-release agent after 28 and 35 days of S. cercariae infection.

Fig 8

Study workflow. (A) The schematic diagram for different agents, including niclosamide sustained-release agents, was applied to the abdomen in vitro before 4–72 h of infection. (B) The schematic diagram for different agents, including S-VCL-S3 recombinant collagen hydrogel and praziquantel sustained-release agents, was administered subcutaneously before or at 4 or 24 h of infection. (C) The schematic diagram for different agents, including S-VCL-S3 recombinant collagen hydrogel and praziquantel sustained-release agents, was administered subcutaneously at 28 or 35 days of infection.