Development of tilapia collagen and chitosan composite hydrogels for nanobody delivery

https://doi.org/10.1016/j.colsurfb.2020.111261Get rights and content

Highlights

  • HCC was fabricated by cross-linking of tilapia skin collagen and chitosan.

  • pH of gel and concentration of collagen and chitosan affected HCC’s stability.

  • Nanobodies (2D5 and KPU) could be sustained release from HCC.

  • The cumulative release of 2D5 from HCC was responsive to changes in pH.

  • HCC hydrogel could be a potential scaffold for drug delivery.

Abstract

Recently, injectable hydrogels have shown great potential in cell therapy and drug delivery. They can easily fill in any irregular-shaped defects and remain in desired positions after implantation using minimally invasive strategies. Here, we developed hydrogels prepared from tilapia skin collagen and chitosan (HCC). The residual mass rate of HCC was affected by the pH at the time of preparation, which was 29.1 % at pH 7 in 36 h. By comparison, the residual mass ratios of HCC at pH values of 6 and 5 were only approximately 8.4 % and 0, respectively. In addition, the stability of HCC was also affected by the concentration of these two components. HCC10 catalyzed by 10 mg mL−1 tilapia skin collagen and 10 mg mL−1 chitosan was more stable than HCC5 catalyzed by 5 mg mL−1 tilapia skin collagen and 10 mg mL−1 chitosan; therefore, we studied that ability of HCC10 to deliver two model nanobodies: 2D5 and KPU. As the concentration of nanobodies increased, the cumulative release rate of 2D5 decreased, and the release rate of KPU increased. Meanwhile, the cumulative release rate of 2D5 was the highest (68.3 %) at pH 5.5, followed by pH 6.8 (56.4 %) and 7.4 (28.4 %). However, the cumulative release rates of KPU were similar at pH 5.5 (45.1 %), 6.8 (46.5 %), and 7.4 (44.9 %). HCC is biodegradable, and can facilitate the release nanobodies; thus, HCC could be developed into an intelligent responsive tumor treatment matrix for use in cancer therapy.

Introduction

Hydrogel is a polymer with a three-dimensional network structure formed by the cross-linking of hydrophilic polymers and can absorb a large amount of water without being dissolved by water [1]. Hydrogels can be transformed to resemble the extracellular environment of human tissue, allowing them to be used in medical implants, biosensors, and drug delivery devices [2]. Injectable hydrogels are cross-linked polymer networks with injectable and moldable in situ characteristics [3], and have been widely studied for their applications to drug delivery, tissue engineering, and especially cancer treatment [[4], [5], [6]]. Biopolymers, such as collagen, hyaluronic acid, and chitosan and its derivates are hydrogel-forming substances with desirable properties [7,8], and they can also provide injectable platforms for cancer treatment [9,10].

Type I collagen is the most abundant collagen in the human body and is primarily distributed in the skin, tendons, and bones; it is also the most abundant protein in aquatic product processing waste [11]. Collagen extracted from fish skin and scales has attracted increased attention, as the extraction of collagen from other animal husbandry operations faces controversy over safety. Therefore, fish skins are generally used as the raw material for extracting collagen, which also increases the value of fish byproducts and reduces environmental pollution [12]. However, the thermal stability of fish collagen is a major disadvantage of its use, as such collagen materials can degrade rapidly when implanted into human tissues for clinical applications [13]. Chitosan is primarily derived from chitin and is a linear amino polysaccharide consisting of glucosamine and N-acetyl glucosamine units through linkage with β (1–4) glycosidic bonds [14,15]. Chitosan has been widely used in drug delivery and tissue engineering applications for its peculiar properties, including high absorbability, permeability, film and fiber formation, moisture absorption and retention, biocompatibility, antibacterial activity, and biodegradability [[16], [17], [18]]. However, pure chitosan hydrogels are highly brittle and weakly viscoelastic and have a poor water-holding capacity, limiting the scope of their applications [19]. Previous studies have reported that collagen-chitosan composite hydrogels possess advantages that could permit them to be designed for use as injectable drug delivery platforms [20].

Despite major advances in therapy, cancer continues to be a major health problem that has not been medically solved [21]. Chemotherapy is one of the primary methods for treating cancer, but patients generally experience side effects, such as nausea and vomiting during therapy periods [22]. The treatment of cancer with monoclonal antibodies (mAbs) drugs is a rapidly growing field [23]. Nanobodies®, which refer to the variable domain of the heavy chain of heavy-chain antibody, were originally discovered in camelids. With approximate molecular weights of 12–15 kDa, nanobodies are considered to be the smallest naturally derived antigen-binding fragments [24]. Nanobodies are featured for their ability to deeply penetrate tissue and also exhibit high solubility with super stability when exposed to extreme conditions, such as low and high pH and temperature [[25], [26], [27]]. Moreover, nanobodies possess an additional disulfide bridge linking the complementarity-determining regions (CDRs) of 1 and 3 (CDR1 and CDR3), enabling the formation of an additional peptide loop with increased flexibility for enhanced recognization of a variety of epitopes [[28], [29], [30], [31]], which cannot be recognized by conventional mAbs. Overall, these specific biophysical and biochemical properties and their potential for targeting novel epitopes make nanobodies promising molecules for diagnostic and therapeutic purposes.

However, the small molecular weights of nanobodies are a double-edged sword. Nanobodies are rapidly cleared from the bloodstream through renal elimination because the sizes of nanobodies are below the renal filtration sieve (approximately 60 kDa) [32], leading to a short half-life and decreased efficacy. This problem can usually be regulated by C-terminal peptide extension, such as provided by a Myc-His-tag, a llama long hinge region-His-tag [33], or by fusion to a "fragment crystallizable chain" (Fc chain) [34]. However, these strategies will have more or less adverse effects on the release or action characteristics of nanobodies in vivo. The use of injectable hydrogels to encapsulate nanobodies and achieve sustained release may be a superior strategy for improving the therapeutic efficacy of nanobodies in vivo.

Previously, we isolated a highly specific nanobody (2D5) against carcinoembryonic antigen (CEA) by immunizing an alpaca with CEA at regular intervals, biopanning nanobodies from a phage display library, and expressing it with E. coli BL21 (DE3). We then selected 2D5 and nanobody KPU against programmed death-ligand 1 (PD-L1) to establish two types of purification methods for tag-free nanobodies [35,36]. Considering their functions and physicochemical properties, 2D5 and KPU were selected as model nanobodies to be encapsulated in injectable hydrogels composed of collagen and chitosan and were gradually released to prolong their retention.

Here, nanobodies (2D5 and KPU) were purified by ammonium sulfate precipitation and Capto MMC. Hydrogels prepared from tilapia skin collagen and chitosan (HCC) were fabricated under different pH values (5, 6, and 7) and incubated with collagenase to assess their optimal preparation conditions. Furthermore, the effect of the formulation of HCC on the stability was also investigated with the in vitro collagenase degradation assay. Different concentrations of 2D5 and KPU (2 mg mL−1 and 4 mg mL−1) were encapsulated in the HCC, and their cumulative release behavior was studied. The microstructures of dried composite hydrogels during degradation and nanobody release were observed by scanning electron microscopy (SEM). In addition, the influence of pH on the release behaviors of 2D5 and KPU was evaluated.

Section snippets

Materials

Chitosan (Mw =1350 kDa) was purchased from Laizhou Haili Biological Product Co., Ltd. (Shandong, China). Fresh Nile tilapia were purchased from the Jimo Aoshan Bay seafood market in Qingdao, China. Skin was peeled from the fish, lyophilized, and used as the raw material for collagen extraction. Phosphate-buffered saline (PBS) solution and trypsin were purchased from Solarbio (cat. P1010, Beijing, China). XBridge® Protein BEH SEC 125 Å, 3.5 μm was purchased from Waters Corporation (cat.

Purification of 2D5 and KPU

The isolation and purification methods of the nanobodies 2D5 (against CEA) and KPU (against PD-L1) have been developed by our previous study [35,36]. Specifically, the nanobody (2D5 or KPU) was expressed in the periplasmic space of E. coli and was obtained with the purification method of ammonium sulfate precipitation and Capto MMC [35]. After two purification steps, a single band of 2D5 or KPU was obtained (Fig. 1). The molecular weight of KPU (18 kDa) is greater than that of 2D5 (14 kDa).

Conclusion

HCC hydrogel was successfully fabricated by the cross-linking of tilapia skin collagen and chitosan, which can be used as hydrogels that can encapsulate anticancer drugs. Here, we showed that HCC can encapsulate two representative nanobodies (2D5 and KPU) and achieve their sustained release. The release behavior of these two nanobodies differed substantially because of their different properties. This study provides a foundation for future studies examining the sustained release of

Consent for publication

All authors have provided consent for the manuscript to be published.

Ethics approval and consent to participate

Not applicable.

CRediT authorship contribution statement

Xiying Fan: Conceptualization, Investigation, Visualization, Writing - original draft, Writing - review & editing. Yunlong Liang: Investigation, Formal analysis. Yuting Cui: Investigation, Formal analysis. Fei Li: Investigation, Validation. Yue Sun: Investigation, Visualization. Junqing Yang: Investigation, Visualization. Haipeng Song: Conceptualization, Writing - review & editing. Zixian Bao: Conceptualization, Funding acquisition, Writing - review & editing. Rui Nian: Conceptualization,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Shandong Provincial Natural Science Foundation, China [ZR2018BC027], National Natural Science Foundation of China [grant numbers 21676286] and QIBEBT and Dalian National Laboratory for Clean Energy (DNL), CAS [QIBEBT ZZBS 201807].

References (48)

  • S. Oliveira et al.

    Targeting tumors with nanobodies for cancer imaging and therapy

    J. Control. Release

    (2013)
  • X. Fan et al.

    Non-affinity purification of a nanobody by void-exclusion anion exchange chromatography and multimodal weak cation exchange chromatography

    Sep. Purif. Technol.

    (2019)
  • A.A. El-Rashidy et al.

    Chemical and biological evaluation of Egyptian Nile Tilapia (Oreochromis niloticas) fish scale collagen

    Int. J. Biol. Macromol.

    (2015)
  • A. Acevedo-Fani et al.

    Edible films from essential-oil-loaded nanoemulsions: Physicochemical characterization and antimicrobial properties

    Food Hydrocolloid.

    (2015)
  • A. El-Fiqi et al.

    Collagen hydrogels incorporated with surface-aminated mesoporous nanobioactive glass: Improvement of physicochemical stability and mechanical properties is effective for hard tissue engineering

    Acta Biomater.

    (2013)
  • C. Park et al.

    New method and characterization of self-assembled gelatin–oleic nanoparticles using a desolvation method via carbodiimide/N-hydroxysuccinimide (EDC/NHS) reaction

    Eur. J. Pharm. Biopharm.

    (2015)
  • L. Wang et al.

    Thermogelling chitosan and collagen composite hydrogels initiated with β-glycerophosphate for bone tissue engineering

    Biomaterials

    (2010)
  • S. Tamilmozhi et al.

    Isolation and characterization of acid and pepsin-solubilized collagen from the skin of sailfish (Istiophorus platypterus)

    Food Res. Int.

    (2013)
  • K. Matmaroh et al.

    Characteristics of acid soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus heptacanthus)

    Food Chem.

    (2011)
  • C. Feng et al.

    Chitosan/o-carboxymethyl chitosan nanoparticles for efficient and safe oral anticancer drug delivery: In vitro and in vivo evaluation

    Int. J. Pharm.

    (2013)
  • J. Cabral et al.

    Hydrogels for biomedical applications

    Future Med. Chem.

    (2011)
  • D. Seliktar

    Designing cell-compatible hydrogels for biomedical applications

    Science

    (2012)
  • T.M.D. Le et al.

    Physically crosslinked injectable hydrogels for long-term delivery of oncolytic adenoviruses for cancer treatment

    Biomater. Sci.

    (2019)
  • H.T. Ha et al.

    Injectable chitosan hydrogels for localised cancer therapy

    J. Control. Release

    (2008)
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