Biosynthesis of elastin-like polypeptides and their applications in drug delivery

doi:10.12211/2096-8280.2021-094

Abstract

Abstract:

Elastin-like polypeptides (ELPs) are artificially synthetic peptide polymers inspired by human elastin. ELPs are composed of repeat units of a Val-Pro-Gly-X-Gly, where X can be any amino acid except proline, and they can exhibit different biological functions along with X residue changes. ELPs are thermally responsive and demonstrate lower critical solution temperature phase behavior. They are soluble at temperatures below a characteristic transition temperature (T_t) and reversibly phase separate into an insoluble, coacervate phase above the T_t. Moreover, the phase behavior is retained when the ELP is either genetically fused to peptides or covalently conjugated to small molecules, and this phase behavior can be adjusted through changing X residue and chain length of ELPs. As ELPs are typically produced from synthetic genes, the structure and function of ELPs can be accurately regulated through genetic engineering. The amino acids or peptides with reactive side chains can be incorporated into ELPs through recombination synthesis as well. This precision control over ELP is unmatched by synthetic polymers. Based on these properties, ELPs can be engineered to assemble into unique architecture and used as soluble macromolecular carriers, therapeutic drug depots, hyperthermia-targeted drug carriers and self-assembled micelles. Lastly, as ELPs are derived from natural protein sequences, they show desirable biological properties including excellent biocompatibility, low immunogenicity, and non-toxic effects. Due to these attributes, ELPs have been widely used in biomedical fields including protein expression and purification, in vitro diagnosis, drug delivery and tissue engineering. By focusing primarily on applications of ELPs in drug delivery, this review introduces the design principles, physicochemical properties, biosynthetic methods of ELPs and ELP conjugates, as well as exemplifies representative applications of ELPs in drug delivery, as extending the half-life of drugs, tumor targeted delivery, local delivery, hyperthermia-targeted delivery and sustained released of the drugs in vivo. Challenges and problems faced in this emerging field are discussed at the end of this review.

Key words: elastin like polypeptides, fusion protein, phase transition, biomaterials, drug delivery

摘要：

类弹性蛋白多肽（elastin-like polypeptide，ELP）是一种衍生于天然弹性蛋白，可人工合成的多肽聚合物。ELP具有特殊的温度响应性，它会随温度的变化表现出可逆相转变行为，并且当它与其他小分子或多肽偶联时，该温敏特性可以被充分保留。借助基因工程可以人工合成ELP与ELP融合蛋白，精确调控ELP的结构与功能，在其序列中添加反应性氨基酸或多肽。同时，ELP由天然氨基酸组成，其生物相容性好，易于生物降解，免疫原性低，无毒性作用。基于以上优势，ELP已被广泛应用于蛋白的表达纯化、体外诊断、药物递送和组织工程等生物医药领域。本文结合国内外研究报道，简要介绍了ELP的设计原理、理化特性和生物合成方法，并列举了一些ELP应用于药物递送系统中有代表性的工作，最后总结了该研究领域面临的挑战和问题。

关键词: 类弹性蛋白, 融合蛋白, 相转变, 生物材料, 药物递送

CLC Number:

Zhaoying YANG, Fan ZHANG, Jianwen GUO, Weiping GAO. Biosynthesis of elastin-like polypeptides and their applications in drug delivery[J]. Synthetic Biology Journal, 2022, 3(4): 728-747.

杨兆颖, 张帆, 郭建文, 高卫平. 类弹性蛋白多肽的生物合成及其药物递送应用[J]. 合成生物学, 2022, 3(4): 728-747.

Figures/Tables 11

Fig. 1 Schematic of ELP construction

Fig. 2 Purification of elastin-like polypeptides (ELPs) and ELP fusions by inverse transition cycling

Fig. 3 ELPs applications in extending half-life of drugs(a) Chemically conjugated ELP to small molecule drugs by inclusion of an intervening cleavable linker which releases the free drug intracellularly after endocytic uptake and accumulation in the acidic, enzyme-rich environment of endosomes and lysosomes; (b) Enhanced cellular uptake of ELP conjugates by functionalization of ELP with cell-penetrating peptides; (c) Schematic of ELP genetically fused to peptide and protein drugs; (d) Pharmacokinetics of NtTNF-VHHELP[43]; (e, f) Pharmacometabolic kinetics (e) and in vivo antitumor effificacy (f) after intravenous injections with IFNα-ELP and IFNα[44]

Fig. 4 Depot-forming ELP for drug delivery(a, b) Drugs form a subcutaneous insoluble coacervate upon injection and slowly dissolve from their surface to their core, steadily releasing the therapeutic into circulation; (c) The drug ELP conjugates can get into a tumor through the EPR effect and then be cleaved into free IFNα and ELP(V) by MMP-2 in the tumor, resulting in enhanced tumor penetration and antitumor efficacy[51]; (d) Fluorescence imaging of mice following MTD subcutaneous injection of Cy5-labeled IFNα-MMPS-ELP (V), IFN-MMPS-ELP (A), IFN-ELP (V), and IFNα; (e,f) pharmacokinetics (e) and antitumor efficacy (f) of mice after subcutaneous injections of IFNα-MMPS-ELP(V)、IFNα-MMPS-ELP(A)、IFNα-ELP(V) and IFNα at their MTDs.

Fig. 5 ELP applied to local delivery(Reservoirs were formed in situ after drug injection, reducing diffusion to non-targeted tissues.)

Fig. 6 Schematic of ELPs in thermal targeting(The tumor tissue was locally heated, and the protein-ELP conjugates were phase transformed and enriched at the heating site.)

Fig. 7 (a) ELP micelles formed by diblock ELP, at a temperature between the Tt of the more hydrophobic ELP block (green, Tt 2) and the more hydrophilic ELP (blue, Tt1), the more hydrophobic block transitions and aggregates while the more hydrophilic block remains soluble, leading to self-assembly into micelles; (b) Cryo-TEM micrograph of ELP micelles[70]; (c,d) Pharmacokinetics (c) and antitumor efficacy (d) after intravenous injections with IFNα-ELPdiblock, IFNα-ELP(A), PEGASYS and IFNα [73]

Fig. 8 Schematic of diblock ELP micelles loading Rapamycin

Fig. 9 (a) Schematic of ELP hybrid nanoparticles loading hydrophobic drug molecules. Hydrophobic drugs are loaded in the core; (b) Synthetic route of LHRH-ELP-DOX nanoconjugates; (c) Cryo-TEM images of LHRH-ELP2-DOX and ELP2-DOX; (d) Pharmacokinetics of LHRH-ELP2-DOX in a DOX-resistant breast tumor mouse model; (e) Tumor volume changes after LHRH-ELP-DOX plus HIFU treatment [78]

Fig. 10 (a) Schematic of ELP hybrid nanoparticles loading hydrophilic drug molecules where hydrophobic sequences act as the core of the assembly when assembled and hydrophilic drugs are loaded within the hydrophobic core; (b) Cryo-TEM micrograph of ATBP-GEM conjugate; (c) AFM image of ATBP-GEM nanoparticles; (d,e) ATBP-GEM delayed the tumor growth (d) and improved the cumulative survival (e) of mice [80]

Tab. 1 Applications of ELP in drug delivery

药物递送策略	应用	ELP药物	ELP序列信息	文献
延长药物体内循环半衰期	髓样乳腺癌	SynB1-ELP-DOX	(VPGXG)₁₅₀ X=V₅G₃A₂	[30]
	感染性休克	^NtTNF-V_HH_ELP	(VPGXG)₁₀₀ X=V₅G₃A₂	[43]
	淋巴瘤	IFN-ELP	(VPGXG)₉₀ X=V₅G₃A₂	[44]
药物储库	2型糖尿病	(GLP-1)-ELP	(GVGVP)₁₂₀	[47]
	卵巢癌和黑色素瘤	IFN-ELP	(VPGVG)₉₀	[49]
	胶质母细胞瘤	IFN-ELP和替莫唑胺联合用药	(VPGVG)₉₀	[50]
	黑色素瘤和卵巢癌	IFN-MMPS-ELP	(VPGVG)₉₀	[51]
	创伤后关节炎	xELP[IL-1Ra]	VPGKG(VPGVG)_16～102	[53]
	神经炎症	ELP-curcumin	[VPGXG] _L_=60,80,160; X = V/I/E [1∶3∶1]	[59]
	骨损伤	rhBMP-2-ELP	(VPGVG)₄₀[(VPGVG)₂(VPGCG)(VPGVG)₂]₂	[62]
热靶向治疗	卵巢癌、宫颈癌、人源咽鳞癌	ELP1	(VPGXG)₁₅₀ X=V₅G₃A₂	[66]
两亲性自组装体	卵巢癌	IFNα-ELP_diblock	ELP(A)₄₈-ELP(V)₄₈	[73]
两亲性自组装体	乳腺癌	FKBP-ELP	G(Val-Pro-Gly-Ile-Gly)₄₈(Val-Pro-Gly-Ser-Gly)₄₈Y	[75]
混合组装体	乳腺癌	LHRH-ELP-DOX	(VPGXG)₁₆₀ X=V₁A₈G₇	[78]
	结肠癌	ELP-(YG)₆-(CGG)₈-GEM	(VPGAG)₁₆₀	[80]
	乳腺癌	DOX_ENC,M-ELP_90A,120	(VPGXG)₁₂₀ X=A₉V₁	[81]
	黑色素瘤	DOX/PPy-ELP-F3	(XGVPG)₁₆₀	[82]
	黑色素瘤、颈瘤、膀胱癌	ELP-AuNP	(VPGVG)₆₀	[89]
ELP水凝胶	骨再生	CDEc	(VPGVG)₁₂₀	[95]

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