Sericin and fibroin nanoparticles—natural product for cancer therapy: a comprehensive review

✅ 全文

丝胶和丝素蛋白纳米颗粒——用于癌症疗法的天然产物:综述

作者 Mehreen Elahi; Shaukat Ali; Hafiz Muhammad Tahir; Rabia Mushtaq; Muhammad Farooq Bhatti 期刊 International Journal of Polymeric Materials 发表日期 2020 ISSN 0091-4037 DOI 10.1080/00914037.2019.1706515 类型 原创研究 (Original Research)

📄 中文摘要 Chinese Abstract

中文
癌症仍然是全球第二大死因,每年约有260万新发病例。化疗等传统治疗方法受限于高毒性、缺乏特异性以及耐药性的产生。因此,对具有生物相容性、可生物降解且毒性较低的药物递送系统的需求日益增长。蚕丝是来源于家蚕(*Bombyx mori*)的天然蛋白质,主要由两种蛋白——丝素蛋白和丝胶蛋白组成,因其良好的生物相容性、可生物降解性、低免疫原性以及能够形成具有增强渗透和滞留(EPR)效应的纳米颗粒等优良特性,已成为癌症治疗领域极具前景的生物材料。

📋 英文结构化总结 English Structured Summary

全文整理

EN

Background:

Cancer remains the second leading cause of death worldwide, with approximately 2.6 million new cases annually. Conventional treatments like chemotherapy are limited by high toxicity, lack of specificity, and development of drug resistance. There is a growing need for biocompatible, biodegradable, and less toxic drug delivery systems. Silk, a natural protein from silkworms (*Bombyx mori*), consists of two main proteins—fibroin and sericin—that have emerged as promising biomaterials for cancer therapy due to their favorable properties including biocompatibility, biodegradability, low immunogenicity, and ability to form nanoparticles with enhanced permeability and retention (EPR) effect.

Methods:

N/A – Review article. This comprehensive review synthesizes existing literature on silk-based nanoparticles derived from fibroin and sericin for cancer therapy. It covers studies involving nanoparticle fabrication techniques (e.g., desolvation, self-assembly, supercritical fluid technology), drug loading strategies, in vitro and in vivo evaluations, and mechanisms of cellular uptake and tumor targeting. The review focuses on preclinical research related to breast, pancreatic, and colorectal cancers, among others.

Results:

Silk fibroin nanoparticles demonstrated high drug-loading capacity, controlled release, and effective tumor targeting via the EPR effect. For example, curcumin-loaded silk fibroin nanoparticles showed enhanced intracellular uptake and apoptosis in breast cancer cells. Similarly, celastrol- and triptolide-loaded silk fibroin nanoparticles inhibited pancreatic cancer cell proliferation and induced apoptosis. Indocyanine green (ICG)-encapsulated silk fibroin nanoparticles enabled pH-responsive photothermal therapy under near-infrared light, effectively destroying cancer cells. Sericin-based nanoparticles, including those conjugated with PEG or PBLG, exhibited improved stability, prolonged circulation, and pH-triggered drug release. Folate-functionalized sericin nanoparticles actively targeted cancer cells overexpressing folate receptors, enhancing doxorubicin delivery and efficacy while reducing systemic toxicity.

Data Summary:

Nanoparticle sizes ranged from 80 nm to over 1000 nm depending on preparation method; for instance, sericin-regulated calcium phosphate nanoparticles were ~80 nm, while some fibroin particles reached up to 10 µm via emulsification. Drug entrapment efficiencies were notably high—curcumin-loaded silk fibroin nanoparticles achieved significant loading due to hydrophobic interactions. In vitro studies reported strong cytotoxicity against MCF-7, PANC-1, MIA PaCa-2, and Caco-2 cancer cell lines, with reduced effects on normal fibroblasts. In vivo animal models showed marked tumor regression following treatment with binary drug-loaded silk nanoparticles. Photothermal studies confirmed effective heat generation by ICG-SF nanoparticles under 808 nm NIR irradiation, leading to cancer cell destruction.

Conclusions:

Silk-based nanoparticles—derived from both fibroin and sericin—represent a versatile, eco-friendly, and cost-effective platform for targeted cancer therapy and drug delivery. Their biocompatibility, biodegradability, and ability to be functionalized for active targeting make them ideal candidates for overcoming limitations of conventional chemotherapy. These nanoparticles enhance drug solubility, enable stimuli-responsive release (e.g., pH or light), and improve therapeutic indices while minimizing off-target toxicity.

Practical Significance:

Silk-derived nanoparticles hold significant translational potential for clinical oncology, offering scalable, low-cost nanocarriers for precision cancer medicine. Their FDA-approved status and natural origin support future development into commercially viable, safe, and effective drug delivery systems for treating various cancers, particularly where current therapies face challenges of resistance and toxicity.

📋 中文结构化总结 Chinese Structured Summary

中文

背景:

癌症仍然是全球第二大死因,每年约有260万新发病例。化疗等传统治疗方法受限于高毒性、缺乏特异性以及耐药性的产生。因此,对具有生物相容性、可生物降解且毒性较低的药物递送系统的需求日益增长。蚕丝是来源于家蚕(*Bombyx mori*)的天然蛋白质,主要由两种蛋白——丝素蛋白和丝胶蛋白组成,因其良好的生物相容性、可生物降解性、低免疫原性以及能够形成具有增强渗透和滞留(EPR)效应的纳米颗粒等优良特性,已成为癌症治疗领域极具前景的生物材料。

方法:

不适用——综述文章。本综述系统整合了关于来源于丝素蛋白和丝胶蛋白的蚕丝基纳米颗粒在癌症治疗中的现有文献研究。内容涵盖纳米颗粒制备技术(如去溶剂化法、自组装法、超临界流体技术)、药物负载策略、体内外评价以及细胞摄取和肿瘤靶向机制等方面的研究。本综述重点关注乳腺癌、胰腺癌和结直肠癌等癌症的临床前研究。

结果:

丝素蛋白纳米颗粒表现出高载药量、可控释放以及通过EPR效应实现的有效肿瘤靶向能力。例如,负载姜黄素的丝素蛋白纳米颗粒在乳腺癌细胞中显示出增强的细胞摄取和促凋亡作用。同样,负载雷公藤红素和雷公藤甲素的丝素蛋白纳米颗粒可抑制胰腺癌细胞增殖并诱导细胞凋亡。包载吲哚菁绿(ICG)的丝素蛋白纳米颗粒在近红外光照射下实现了pH响应型光热疗法,有效杀伤癌细胞。丝胶基纳米颗粒(包括与PEG或PBLG偶联的纳米颗粒)表现出改善的稳定性、延长的血液循环时间以及pH触发的药物释放特性。叶酸功能化的丝胶纳米颗粒可主动靶向过表达叶酸受体的癌细胞,增强阿霉素的递送和疗效,同时降低全身毒性。

数据总结:

纳米颗粒的粒径范围从80纳米到超过1000纳米不等,具体取决于制备方法;例如,丝胶调控的磷酸钙纳米颗粒约为80纳米,而某些通过乳化法制备的丝素蛋白颗粒可达10微米。药物包封率显著较高——负载姜黄素的丝素蛋白纳米颗粒由于疏水相互作用实现了显著的载药量。体外研究表明,其对MCF-7、PANC-1、MIA PaCa-2和Caco-2癌细胞系具有强细胞毒性,而对正常成纤维细胞的影响较小。体内动物模型显示,经双药负载的蚕丝纳米颗粒治疗后肿瘤明显消退。光热研究证实,ICG-SF纳米颗粒在808纳米近红外光照射下能有效产热,导致癌细胞死亡。

结论:

蚕丝基纳米颗粒——来源于丝素蛋白和丝胶蛋白——代表了一种多功能、环保且具有成本效益的靶向癌症治疗和药物递送平台。其生物相容性、可生物降解性以及可功能化实现主动靶向的能力,使其成为克服传统化疗局限性的理想候选材料。这些纳米颗粒可提高药物溶解度,实现刺激响应型释放(如pH或光响应),并提高治疗指数,同时最大限度地减少脱靶毒性。

实践意义:

蚕丝来源的纳米颗粒在临床肿瘤学领域具有重要的转化潜力,为精准癌症医学提供了可扩展、低成本的纳米载体。其FDA批准状态和天然来源支持其未来开发为商业上可行、安全且有效的药物递送系统,用于治疗各种癌症,特别是在当前疗法面临耐药性和毒性挑战的领域。

📖 英文全文 English Full Text

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International Journal of Polymeric Materials and Polymeric Biomaterials

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Sericin and fibroin nanoparticles—natural product for cancer therapy: a comprehensive review

Mehreen Elahi, Shaukat Ali, Hafiz Muhammad Tahir, Rabia Mushtaq &

Muhammad Farooq Bhatti To cite this article: Mehreen Elahi, Shaukat Ali, Hafiz Muhammad Tahir, Rabia Mushtaq &

Muhammad Farooq Bhatti (2020): Sericin and fibroin nanoparticles—natural product for cancer therapy: a comprehensive review, International Journal of Polymeric Materials and Polymeric

Biomaterials, DOI: 10.1080/00914037.2019.1706515 To link to this article: https://doi.org/10.1080/00914037.2019.1706515

Published online: 19 Feb 2020.

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View Crossmark data Sericin and fibroin nanoparticles—natural product for cancer therapy: a comprehensive review

Mehreen Elahia, Shaukat Alia, Hafiz Muhammad Tahira, Rabia Mushtaqa, and Muhammad Farooq Bhattia,b aDepartment of Zoology, Government College University, Lahore, Pakistan; bSericulture Wing, Forest Department, Lahore, Pakistan

ABSTRACT Silk, a natural compound of silkworm contains two proteins, sericin and fibroin. These proteins can be conjugated with other compounds to form silk-derived nanoparticles. The biomedical applications of silk-based nanoparticles for drug delivery and cancer treatment are arising as they are biocompatible, biodegradable, have enhanced permeability and retention effect and less tox- icity. Nevertheless, not a single review of literature is present that could describe the anticancer potential of silk derived nanoparticles. In this review, we describe the (i) comprehensive note on fibroin and sericin based nanoparticles (ii) anticancer mechanistic accompanied by biomedical applications in diagnosis, imaging, and drug delivery.

GRAPHICAL ABSTRACT ARTICLE HISTORY Received 4 October 2019

Accepted 5 December 2019 KEYWORDS Biocompatible; cancer treatment; biodegradable; drug delivery; fibroin; sericin; silk-based nanoparticles

CONTACT Shaukat Ali dr.shaukatali@gcu.edu.pk Department of Zoology, Government College University, Lahore, 54000-Lahore, Pakistan.

Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpom.

 2020 Taylor & Francis Group, LLC INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS https://doi.org/10.1080/00914037.2019.1706515

1. Introduction Even though great improvement has been done for the advancement in the progression and formulation of new drugs and various therapeutic approaches for cancer but still cancer continues to be the second most leading reason for death worldwide. The yearly occurrence of cancer is nearly about 2.6 million cases per year[1,2]. Necessarily, there is a crucial requirement of global measures to accompany the advantages of novel treatments in the industrialized areas and take appropriate actions to bring about the already pre- sent treatments of cancer available in the underdeveloped and developed sectors[3]. The latest studies have made con- siderable advancement in the identification of underlying mechanisms and particular causes for various forms of can- cer. Many of the cancer therapies are being used such as photodynamic therapy, radiation therapy, chemotherapy, vaccinations, stem cell transplantations and also conjugation of these treatments. Inopportunely, the majority of the trad- itional treatments are expensive and also cause harmful reac- tions[4,5]. Mainly the chemotherapy treatment causes high toxicity to the cells including both the cancerous and normal cells and thus limits its applications clinically[6].

Furthermore, another issue is the resistance of cancerous cells to these chemotherapeutic means[7], hence it becomes important to find such anti-tumor agents with biocompati- bility, biodegradability and less toxicity to cells. A drug gives optimum results when its dispersal profile is both definitive and regulated and finally, such a delivery system is required that is compatible and offers adjustability such that of the silk proteins[8]. Thus, research should be done on the gener- ation of economically suitable and efficient treatment choices that not only target the specified cancers but also does not cause any harm to the normal healthy tissues[9].

A delivery system for drugs contains a carrier containing active drug component in dispersed, dissolved or encapsu- lated on which the functional component is joined or adsorbed[10]. Drug carrier molecules are very important in drug delivery and could be administered in various forms of drugs released systems including microcapsules, nanopar- ticles, pills, microspheres, emulsions and many more.

Nanoparticles among them have fascinated much of the research because of their capability of being utilized as an effectual transporter of drug supporting drug efficiency[11].

These nanoparticles also offer new prospects to overpower the traditional methods of delivery restrictions concerning drugs[12]. Nowadays these new delivery systems utilizing nanoparticles are of great attention because this helps tar- geted introduction to the specific sites of the small bioactive molecules and drugs[13].

Recently, protein-built nanocarriers are gaining import- ance because of their biodegradability, biocompatibility, great nutritional value and less toxicity to the cells. These proteins derived nanocarriers also display great binding affinities letting remarkable uptake by the cells[14]. The scheme and formulation of effective delivery systems of a drug are central to the biomedical field[12]. Nanocarriers also offer increased anti-tumor efficiency showing minimal side effects due to their features including increased EPR effect, time in blood circulation and uptake into the cell actively[15,16]. Biopolymers varying from bioactive polymers such as heparin to the large molecular weight drug carriers including chitosan, dextran and alginates and many multiple function molecules[17,18]. One of these naturally occurring biopolymers is silk, which is being utilized in suturing and is approved by the FDA for application in humans for carry- ing the load. However, currently, silk has been rising as a prominent drug delivery biopolymer[19].

The silk originally has two key proteins including fibroin and sericin[20,21]. Silk is formed by the silkworm mainly

Bombyx mori. The sericin component is the soluble part of the cocoon and encloses and retains two filaments of fibroin together in the silk thread present in the silk cocoon[22]. The fibroin component is the insoluble protein having massive hydrophobic domains consisting of b-pleated sheets in an antiparallel direction[23]. Consequently, the requirement for biodegradable and biocompatible materials displays the ris- ing interest of silk protein components in a huge field of biomedical[22]. Sericin protein can be utilized in the drug delivery application because of its pH responsiveness and reactivity that helps in binding with other molecules and thus enabling the production of small molecules[24]. Silk fibroin component has also high binding capacity with sev- eral kinds of drugs, sample preparation requirements, con- trolled release of drug characteristics.

Both of the components of the silk have exceptional biodegradability, biocompatibility and very little or no immunogenicity[11].

Currently, nanotechnology can participate significantly in drug delivery, diagnosis and cancer treatment. Anticipating the lack of review on the usage of silk-based nanoparticles as a promising agent for cancer therapy involving the drug delivery applications, we designed this thorough review art- icle. We for the first time explained the anticipated drug delivery and anticancer activity of the silk fibroin and sericin based nanoparticles with innovative dialogue on the key advances of utilizing these nanoparticles to the clinical stage for cancer therapy (Figure 1).

2. Biomaterials The widely used pharmacological anticancer agents impart many adverse effects on the normal cells along with the can- cer tissue, so chemotherapy has many severe side effects[25].

Therefore, natural products such as biomaterials play a sig- nificant part in tissue engineering, medicine of regeneration and in the delivery of drugs[26].

3. Nanotechnology Nanotechnology has an immense role in many fields as one of its applications is nanomedicine utilized in the field of medicine. This has aided in the advancement of therapeutic carriers in the form of nanoparticles. On account of the nanoparticles enhanced the ratio of surface to volume as compared to the bulky substances impart them with fasci- nating properties like augmented mechanical strength[27].

The nanoparticle drug carriers are developed through

2 M. ELAHI ET AL. various routes depending on the application. A wide variety of materials are designed but the mutual goal of all is to explain the transfer of the drug and its enhanced bioavail- ability toward the directed cells and endorsing the response with minimized side effects. The delivery of drugs toward tumors is aggravated by harmfulness to the normal cells along with the little absorption because of less retainment of drugs by the cancerous cells at the site of the tumor[28].

Many processes like differentiation, the proliferation of cell and tissue regeneration depends on the connections between the cells and the surfaces of biomaterials[29].

The tumor microenvironment embraces cells, blood ves- sels, extracellular matrix and signaling molecules[30].

Carcinomas are the most frequent tumor types (85%). In most of the cases, these are non-vascularized and does not allow the tumor to grow greater than 2 mm and does not make new blood vessels for their feeding[31]. Angiogenesis is dysregulated for feeding the tumor cells, and thus the vessels formed vary from that of the normal tissue. Presently, nanocarriers are used efficiently for targeted therapy of can- cer and transport the desired drug component which can pass over the vasculature. Some of the vehicles of nanocar- rier having a diameter of 20–300 nm are being established for drug or medicine and many more therapeutic particle transports. These treatments are directed to extravasate selectively over the vasculature of cancer via EPR (Enhanced

Permeability and Retention) effect[32].

The Nanomedicine and Drug Delivery Symposium (nano-DDSs) drugs remain a capable approach to converse the resistance of drug, for its effective intracellular delivery.

In the meantime, earlier findings have described that the nanoparticles could be carried into compartments of endo- somes like lysosomes which are located in the nuclear per- iphery regions substantially further than the transporters of

P-gp on the membrane. This benefit of location consents release of drugs inside these compartments of endosomes to minimize efflux pumping, subsequently, the microenviron- ment of lysosomes and tumor cells with low pH might offer

Figure 1. Silk-based nanoparticles have various biomedical applications like in tissue engineering, diagnosis, imaging, drug delivery and cancer therapy.

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3 a strategy meant for pH-stimuli receptiveness[33]. The nano- DDSs can efficiently enter the tumor site by EPR effect during the circulation of blood in comparison to chemother- apeutic vehicles which are free[34–38]. Under these condi- tions, the drugs warped inside the nanocarriers might directly cross the cell membrane, and released toward the nucleus, therefore induced the death of most cancer cells.

Thus, nanomedicines that are taken up and released in this manner could have a more effective anti-tumor effect[39].

4. Silk fibroin The insoluble protein called silk fibroin (SF) has massive hydrophobic domains consisting of b-pleated sheets in an antiparallel direction and could be simply separated as bio- materials excluding sericin[23]. It is produced from spiders and silkworms including the Bombyx mori and Bombyx mandarins larvae along with other genera of the moth, for example, Samia, Cricula, Gonometa, and Antheraea, and many other insect’s genera. The silk fibroin primary struc- ture is categorized as a naturally occurring unit of copoly- mers which comprise of hydrophobic chunks having repeated small side-chain of amino acids like alanine and glycine and primarily comprising of repeated order of (Gly–Ser–Gly–Ala–Gly–Ala)n amino acid. The original con- dition has two key proteins including fibroin and sericin, where sericin is glue-like material coating fibroin’s two brins which are singular filaments[20,21]. This material is extremely appropriate because of its low inflammatory or immune response and promising biological response features[40]. Silk

Fibroin-based biomaterials are being explored as fibers, par- ticles, files, and scaffolds[41–44], and in practices of neural, hydrogels, bone, skin, vascular and cartilage tissue recov- ery[45–48]. Gradually, SF is being utilized in many other zones of biomedical science, because of its properties and novel knowledge about its processing such as elasticity, mechanical strength, controllable biodegradability and bio- compatibility[49]. These characteristics of SF are mainly beneficial for tissue engineering[29].

4.1. Silk fibroin nanoparticles Breast cancer has been a constant source of growing disease and death in women. Shortly, the chemotherapy of breast cancer will encompass the use of drug delivery vehicles with biocompatible high cell-targeting capacity such as silk fibroin for the betterment of these problems. The usage of silk fibroin (SF) nanoparticles for the transfer of cytotoxic drugs offers optimal entrapment, specificity, enhanced thera- peutic index, and maximum toxicity of cells of breast cancer with minimum or no harm to nearby normal cells. Silk fibroin is loaded with suitable chemotherapeutic drugs (e.g., carboplatin). Silk Fibroin has been described in numerous studies as a very valuable tool in precise passive or active delivery of a drug to targeted cancer cells, hence confirming maximal devastation and minimal harm to normal nearby cells, which enhances the drug efficacy and reduces the sys- temic toxicity[50] (Figure 2).

4.2. Silk fibroin modified chitosan nanoparticles (SF-CSNPs)

Silk fibroin modified chitosan nanoparticles (SF-CSNPs) obtained more consideration as carriers of drug or medicine on account of their low toxicity, better stability, mild prepar- ation procedures, and also offer versatile administration routes[51]. The SF-CSNPs are appropriate for the chemother- apeutic drug delivery for cancer treatment because of their

EPR effect on tumor cell surfaces. The SF-CSNPs are accu- mulated by the hydrophobic interactions around the defected cells and tissues. Additionally, numerous physico- chemical parameters of SF-CSNPs are essential to be found as favorable, as its capability to traverse the biological block- ades, to defend macromolecules breakdown and to carry the complex to the targeted location[52].

Yang et al.[29] confirmed the biological effects of uptake of SF-CSNP by the liver cells. They studied various proteins that were involved in the ubiquitin-proteasome pathway and verified that SF-CSNP might be engaged in the propagation and persistence of liver cancer cells[29].

4.3. Self-assembled silk fibroin nanoparticles The exclusive characteristics of nanoparticles can overwhelm the restrictions of using the micro molecules as representa- tives of therapeutics in biomedical applications[53]. For instance, the drug uptake can be improved by the EPR effect[54], which can surge the deposition of drugs in tar- geted cancer containing tissues and decrease the drug resist- ance by efflux pump–mediation. Moreover, the half-life and solubility of small molecular medicines or drugs could be enhanced with controllable releasing actions along with encapsulation by nanoparticles. Curcumin is a naturally existing compound derivative of turmeric with diphenolic groups and is extensively used in radio-sensitizing and chemotherapy of cancer[55–57] and other fields of biomed- ical[58–60] with nearly no toxicity[60,61], and act as a capable material in the clinical examinations for the cancer treat- ment. But, due to curcumin’s poor solubility in the physio- logical environment, it frequently displays little uptake or absorption inside the gut[62]. Lately, curcumin was encapsu- lated with the SF nanoparticles and directed to the cancer cells specific to breast and showed the highest entrapment, intracellular uptake, and regulated release[28]. Furthermore, it has been discovered that two or more drugs, when admin- istered concurrently, is also significant in the treatment of cancer. Owing to silk fibroin biocompatible and biodegrad- able features it is thought to be a promising agent in bio- medical fields[43,63–66]. SF molecules as a drug carrier have controlled breakdown in vivo and in vitro, and could be controlled through altering its molecular weight, size, level of cross-linking, and crystallinity[42,44]. Many features of SF molecules make it an excellent agent as a drug carrier. One is that its hydrophobic residues of amino acid, such as gly- cine, tyrosine, and alanine boost the curcumin entrapment and 5- fluorouracil (5-FU) by p–p wrapping and hydropho- bic interface enhance the loading efficiency of binary trans- porter of drugs. Besides, the hydrophilic residues of amino

4 M. ELAHI ET AL. acid, such as glutamate, aspartic acid, and serine enhance their solubility in water and cause the development of nano- particles in the aqueous solutions[45]. One of the studies cre- ated

SF nanoparticles acting as a biodegradable and biocompatible transporter for the efficient loading and trans- ferring of binary drugs 5-FU and curcumin and exhibited an exceptional in vivo and in vitro anticancer activity. It is being discovered that treatment with such kind of nanodrug could augment the amount of ROS (reactive oxygen species) which induces in vitro cancer cells apoptosis. Animal researches have revealed that tumors can be markedly decreased after being inoculated with the nanoparticles entrapping the drug. This demonstrated the future possibil- ity of breast cancer treatment with binary drugs loaded with nanoparticles[46].

4.4. Celastrol and triptolide loaded silk fibroin nanoparticles

Pancreatic cancer is one of the fatal diseases with a 5% sur- vival rate for 5-year. Despite many treatment options, its diagnosis is extremely poor. Hence, different therapies are employed to fight against this fatal disease such as using a combination of drugs. Celastrol (CL) and Triptolide (TPL)

Figure 2. Proposed mechanism of the role of silk fibroin-based nanoparticles in cancer therapy. Silk fibroin-based nanoparticles have enhanced drug loading cap- acity and this conjugate of silk fibroin nanoparticles loaded with drug moves through blood circulation and reach targeted site through the EPR effect and internal- ized through endocytosis into the tumor cell. The lysosomes carry them to the nucleus where the drug is released because of acidic pH and causes DNA impairment and induce apoptosis.

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5 are the two main compounds used in Chinese traditional medicine and have a wide range of bioactivities, one of which is the anticancer activity[48,49,51]. As silk fibroin is a natural polymer with various unique features are used as the best carrier material. One of the studies prepared CL and

TPL loaded SF nanoparticles (CL-SFNPs and TPL-SFNPs) through an improved desolvation method[47]. TPL revealed to be an efficient stimulator for the halt of the apoptosis and cell cycle in numerous categories of cancers like breast cancer, pancreatic cancer, and lung cancer[14,52,67,68]. It could efficiently reduce the in vitro viability of the pancre- atic cancer cells, as well as decrease the metastasis and growth of in vivo tumors[49,52,69].

Celastrol represses the incursion of pancreatic cancerous cells by downregulation of the chemokine receptor CXCR4 expression[70]. Nevertheless, due to their less solubility in water and severe toxicity, CL and TPL could not be employed in the clinics systemically. Hence, there was a necessity for the development of the alternative CL and TPL formulations for clinical use. Ding et al.[71] developed and characterized the CL and TPL loaded SF nanoparticles (CL- SFNPs and TPL-SFNPs) separately. This not only overcome the drawback of hydrophobicity but too enabled the passive accumulation of CL and TPL in the cancerous tissues cen- tered on the optimal dose and administration and the EPR effect. After the formulation, characterization and optimiza- tion of CL-SFPNs and TPL-SFNPs were evaluated. The SF nanoparticles cellular uptake behavior was inspected in MIA

PANC-1 and PaCA-2 cells with RITC (Rhodamine-B- Isothiocyanate) used as a fluorescent probe was established by the flow cytometer and confocal microscopy.

The advantage of the collective treatment of CL-SFNPs admixed

TPL-SFNPs were confirmed by cell apoptosis and anti-pro- liferation detection in the pancreatic cell lines of humans such as PANC-1 and MIA PaCA-2[47].

4.5. Indocyanine green encapsulated silk fibroin nanoparticles

In the earlier few decades, numerous therapeutic approaches using light were the probable alternatives to the traditional methods in the arena of healthcare for their striking proper- ties incorporating biocompatibility, minimum invasiveness, great selectivity, and favored photosensitizers localization ensuing negligible adverse effects[72–74]. Photothermal ther- apy (PTT) was the one amid them and utilizes photosensi- tizers, such as PTT mediators that function by converting absorbed energy of light at a specificwindow of wavelength to heat to show the therapeutic actions[75–79]. Lately, there has been an eminent attention in the improvement of NIR (near-infrared) triggered thermotherapy hostile to cancer because of its outstanding tissue penetration, minimal inva- siveness and high efficacy of

NIR (>750 nm)[80,81].

Indocyanine green (ICG) is a NIR fluorescent dye certi- fiedby the US Food and Drug Administration (FDA) and is used in numerous biomedical applications. ICG is particu- larly important in diagnosis, imaging, and therapeutics in the biomedical fields because of its striking features including excellent biocompatibility and important light-to- heat transformation efficiency among others[75,76,82,83]. But,

ICG still has few limitations including poor stability in aque- ous medium and absorption in the intestine, accumulation depending on the concentration in vivo ensuing poor intra- body recirculation, susceptible to photobleaching, and the absence of specificity for the target[77,82,84]. To overcome these problems, ICG being transported using various nano- carriers with enhanced stability and directing efficiency including polymeric carriers (PCs), layered double hydrox- ides (LDHs), liposomes and mesoporous silica nanoparticles (MSNs) among others[75,76,83,85–87]. The heavy chain of SF is noticeably large containing

C-terminal and N-terminal hydrophilic areas and twelve greatly recurring regions of gly- cine-alanine rich flanked by the interior hydrophilic zones[88]. The N-terminal area in this structure of fibroin (FibNT) undertakes a pH-sensitive conformational modifica- tion at below 6.0 pH to b-sheets from the random coils and results in the transfer of loaded therapeutic vehicle[88,89].

Many researches have reported the formation of silk fibroin nanoparticles including emulsification (>6 lm), poly (vinyl alcohol) blends (300 nm–10 lm), salting leaching (486–1200 nm), capillary microdot printing (25–140 nm), supercritical CO2 (50–300 nm)[22,90], and organic solvent precipitation (35–170 nm). A number of researches have observed the capability of SF nanoparticles to capture and release the drugs[12,22,88,89,91–93].

The supercritical fluid (SCF) technology has acquired much attention among them from the investigators in the earlier period for producing the polymer molecules and further therapeutic bio-actives because of its economical character and environmentally benign nature[93–95]. To achieve the production of the multi- purpose dual-triggered devices with desirable biodegradation and high therapeutic efficiency,

ICG-encapsulated silk fibroin (ICG-SF) nanoparticles were formulated by SCF technology. These ICG-SF nanoparticles formed by using this method has provided exceptional photothermal con- stancy, ICG liberation from SF by pH-responsiveness par- ticularly in the acidic tumor microenvironment, and its significant initiation with NIR light at 808 nm substantially improved PTT efficacy. Photothermal in vivo and in vitro trials have revealed that these ICG-SF nanoparticles have the capability of destroying the cancerous cells simply in the light-triggered hyperthermia. These outcomes together have proposed that ICG-SF nanoparticles formed by the SCF method gave rise to enhanced PTT efficiency and might be employed a s a favorable material in the delivery system for continuous therapy of cancer[96].

5. Silk sericin The sericin component of silk is a naturally occurring polymer that is hydrophilic and mainly formed by the insects of family

Saturniidae and Bombycidae. It is formed through epithelial cells and then collected in the generalized silk glands lumen of developed larvae of 5th instar. Then discharged from the silk gland middle and posterior segment[67]. Sericin is about

15–20% of the dry cocoon weight and adheres to the filaments

6 M. ELAHI ET AL. of fibroin of silk cocoon together. Sericin from silk is obtained during the silk fibers degumming process and is usually removed as waste material in the industry of textile. Now a days it imparts fascinating applications in the engineering of tissue and biomedical fields[14,68]. Sericin protein contains chiefly amino-acids for instance glycine (16%), serine (40%), aspartic acid, glutamic acid, tyrosine and threonine. It com- prises of polar side chains composed up of amino, carboxyl, and hydroxyl groups that allow easy copolymerization, cross- linking, and combination with more polymers to formulate better biodegradable substances[69,70,90]. The physicochemical attributes of sericin primarily depend on the way of sericin iso- lation and the families of the silkworm and this affects its func- tional features and mark sericin as a probable biocompatible material for various biomedical applications[22] (Figure 3).

5.1. Silk sericin self-assembled/poloxamer nanoparticles

In current times self-assembled micellar nanoparticles have been effectively working for directed drug delivery purposes in tissue engineering. In Mandal and Kundu[97] review article, silk protein sericin from the Antheraea mylitta non-mulberry

Tasar silk cocoons obtained from tropical region were mixed with pluronic F-87 and F-127 along with solvents to attain self-assembled nanostructures in the form of micelles that have the capability of carrying both hydrophobic (anticancer drug paclitaxel) drugs and hydrophilic (FITC-inulin). The quick intake of these units into the cells was experimented in in vitro researches using the MCF-7 cells of breast cancer. In vitro cells toxicity examination utilizing the nanoparticles loaded with paclitaxel against the breast cancer cells exhibited potential consequences compared to the paclitaxel drugs alone. Encapsulated drugs with nanoparticle have stimulated apoptosis in the

MCF-7 cells of breast cancer.

Downregulation of Bcl-2 (anti-apoptotic protein), upregula- tion of Bax (pro-apoptotic protein), and cleavage of PARP (regulatory protein) proposed more drug, provoked apoptosis in the cells. The research develops silk protein sericin as a substituted biomaterial for the production of self-built nano- particles in the occurrence of poloxamer for the effective

Figure 3. Proposed mechanism of silk sericin-based nanoparticles in cancer treatment. The diagram shows the silk sericin- nanoparticles loaded with the drug are internalized via clathrin-mediated endocytosis into the cancerous cells and carried through lysosomes into the tumor microenvironment with low pH, releasing the drug and induction of apoptosis through the cleaved caspace-3 pathway.

INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS

7 distribution of both hydrophilic and hydrophobic drugs to the targeted locations. This offers a new aspect to naturally occurring sericin protein in the nanoparticles form for its effective and efficient role as a drug delivery vehicle in tissue engineering and biomedical applications[97].

5.2. Sericin-PEG nanoparticles and sericin-PBLG micelles

There are many materials used as nanocarriers, but the nat- ural polymers are chosen over current years. Among these natural polymers, sericin has obtained great consideration.

Due to its several valuable properties, sericin-based substan- ces are progressively applied in biomedicine and tissue engineering[24,41]. Presently, numerous procedures have been established to make silk centered nanoparticles such as seri- cin-PEG nanoparticles, self-built sericin nanoparticles, or other types of silk or sericin micelles[89,97–106].

Subsequently, sericin is viewed as a new material in the field of the nanocarrier. Synthetic polypeptides are broadly uti- lized in the spheres of biomedical like in the delivery of drugs[107] because of their intrinsic biodegradability and bio- compatibility degradation products. Poly (c-benzyl-L-glutam- ate) (PBLG) formed synthetically has gained consideration, and is being attached to the hydrophilic backbone of poly- saccharide identical to hyaluronic acid[108] or attaching of synthetic hydrophilic polymer like PEG[109] to develop core–shell designed micelles. In these amphiphilic unit poly- mers, the core of PBLG aids as a reservoir for hydrophobic drugs and significantly enhances the stability of therapeutic agents in the blood circulation. Furthermore, PBLG can be degraded into an amino acid such as L-glutamic acid[110].

Sericin-PBLG micelles show high stability with high drug loading capacity, which helps in extended circulation time in the blood. These micelles correspondingly had reliable biocompatibility, negative surface capacity, and suitable size distribution. The nanoparticles can be proficiently interior- ized via clathrin-facilitated endocytosis into the cells. Sericin

PBLG-DOX was carried, and then gathered within the per- ipheral nuclear lysosomes faraway from transmembrane drug pumps, and afterward releasing DOX in the micro- environment with below low pH. The DOX which is released entered directly into the nucleus following DNA impairment and greater anti-cancer effect. The principal mechanism is still to be investigated as an outcome of the pattern of these nanocarriers. The micelles of sericin-PBLG- DOX administration can accomplish an appropriately ele- vated concentration of native drug that augments the DOX chemotherapeutic impact directed toward tumors, hence reducing the unwanted cytotoxic side impacts, together with damage to brain, liver, heart, lungs, kidneys, and spleen.

The micelles of sericin-PBLG-DOX are safe and helpful in the chemotherapeutic drug delivery and thus offer a prob- able technique to reverse the multidrug resistance[39].

5.3. Sericin regulated spherical calcium phosphate nanoparticles

Calcium phosphates (CaPs) are very significant inorganic compounds of biological hard tissues in hydroxyapatite form. They have been employed as best biomaterials in the clinical inquiries because of their exceptional biocompatibil- ity function. The usage of premixed rapidly resting calcium phosphate mixtures for the bone repair can expressively improve the graft properties and cut the surgical time[111–113]. Currently, CaP nanoparticles biomedical appli- cations have spread to many other fields including gene and drug transfer and in vivo imaging reliant on progression in the nanotechnology field[114–117]. CaP has been employed as a transfecting agent for many years and its transfection effi- cacy has been enhanced by altering the size of the particle and executing surface amendments[118–122]. CaP nanopar- ticles offer a unique group of vessels for the delivery of drug as it shows sensitivity to acidic pH in the lysosomes of cell and little immunogenicity. This allows overcoming the prob- lem of drug resistance. These CaP nanoparticles could be charged with a range of molecules comprising siRNA, pDNA and chemotherapeutic drugs[123]. The dispersal along with CaP nanoparticles degradation in vivo is very import- ant for use as in vivo delivery of drug or gene[124].

The CaP nanoparticles passive targeting is still unclear after the intravenous injection and restricts its uses in the gene or drug delivery. Thus, an effective and safe approach for noticing the CaP nanoparticles delivery and tumor tar- geting in vivo is a demanding need now a days[124]. In prior studies, it was revealed that sericin can retain the amorph- ous phase of CaP nanoparticles with greater sensitivity of pH yielding it suitable for the use in the delivery of drugs or genes for the therapy of cancer[125–127]. S-CaP nanoparticles of size 80 nm were formed and tagged by near-infrared dye reagent (DiR) (S-CaP@DiR to inspect the CaP nanoparticles delivery and degradation in vivo, and the emitting NIR fluorescence from DiR offers a shallow way of in vivo imag- ing of non-invasiveness in the real time[128] to clearly detect the dispersal, breakdown or tumor targeting in in vivo which is the main reason of S-CaP nanoparticles manipulated in gene or drug delivery[124].

5.4. Multifunctional sericin nanoparticles Nanocarriers have increased the loading capacity of drugs and can be combined with hydrophobic or hydrophilic drugs. Notably, they are intended to have the capability of targeting the cancer cells actively through nanocarrier-linked ligands that explicitly attach to molecules in the cancer cells.

Natural polymers are of great attention to the use of materi- als for the nanocarriers[129]. The versatile chemical structures of these materials permit them to be suitably functionalized with the stimuli-responsive groups and tumor targeting ligands toward the design of excellent drug delivery sys- tems[130]. Such complexes release materials to the tumor sites through chemical bonds cleavage in the presence of probable external or physiological stimuli, for example, enzymatic activity[131], temperature[132], or pH[133,134] within the microenvironment of the tumor. Sericin is a abundant in polar side chains composed of carboxyl, amino and hydroxyl groups that offer it with increased chemical reactivity and not any immunogenicity[135–138] and has

8 M. ELAHI ET AL. varied bioactivities and excellent biocompatibility with the tissues and cells[139,140]. Hence, several formulations of seri- cin for the applications in biomedical have been discovered, including films[141], hydrogels[142], microparticles[143] and

3 D scaffolds[144]. In the nanocarriers field, two kinds of self- assembled sericin nanoparticles have been described for gene and drug delivery including sericin-poloxamer nano- particles[97] and sericin-PEG[69], and a kind of desolvated sericin nanoparticles[145]. All these stated sericin nanopar- ticles are a deficit in targeting the tumors in a specialized manner. Therefore, multifunctional sericin nanoparticles are a valuable tool for cancer therapy with specific targeting of the tumor and controlled release of drugs[105].

Owing to increased expression of folate receptors in sev- eral cancerous cells of humans[146], Huang et al.[105] pro- duced sericin nanoparticles with the capability of active targeting of tumor then to be favorably endocytosed through receptor-mediated endocytosis by the cancerous cells. In the meantime, endocytosis traffics nanoparticles into the intra- cellular compartments of endo-lysosomes having low pH (acidic). They designed pH sensitive hydrazone bonds to link sericin and DOX, hence bestowing controlled release of drug attribute to the nanoscale delivery approach. This cova- lent linkage helped to decrease the possible leakage of the drug. Conjugates of DOX-sericin could self-assemble to form nanoparticles because of the DOX hydrophobic nature and sericin hydrophilic nature. This approach would enable the successful liberation of DOXintracellularly, which pos- sibly will augment the anti-tumor effect of this system of nanoparticles of sericin. This plan was implemented for the successful fabrication of folate-sericin-DOX (FA-SND) based nanoparticles and was potentially employed asa carrier for drug transferfor the therapy of cancer[105].

5.5. Resveratrol-loaded sericin nanoparticles Resveratrol having the chemical name as trans-3, 5, 40-trihy- droxy-stilbene is a polyphenolic complex that has been recognized an anti-inflammatory[147], anti-carcinogenic[148], and anti-oxidant[149] characteristics which make it an inter- esting bioactive compound in pharmaceuticals. Its anti-can- cerous prospective has been broadly studied and is particularly remarkable, though its practicable uses are restricted by its poor solubility in water, quick degradability and photo-sensitivity[150,151]. One of the studies developed nanocarriers of protein by the modified-desolvation, using a retrieved protein powder of sericin from wastewater.

Manufacture of nanocarrier was carried out via a range of silk protein and concentrations of pluronic stabilizer, allow- ing the choice of optimal requirements for the development of minor sized, spherical, and stable nanoparticles. The silk protein nanoparticles were fabricated with resveratrol (RSV) by precipitation procedure involving no solvent and addition of pluronic surfactant 0.5% (w/v). These RSV-loaded SP nanoparticles effectively repressed the growth of Caco-2 (colorectal adenocarcinoma) cells while showed non-cytotox- icity to the fibroblasts of skin, as shown by the cell viability assays.

SP nanoparticles internalization cellular proved superficial and reliant on incubation time, carrier’s transfec- tion, in vitro outcomes representing continuous discharge of

RSV, and enhancements of the drug after encapsulation indicate their promising applications in the pharmaceutics and therapeutics. Therefore, sericin protein nanoparticles is a favorable attempt to be probable biological nanocarrier for the transport system for drug[152].

6. Conclusion and future perspectives The greater incidence of various types of cancer and its sev- eral treatment limitations such as high cost of drugs, resist- ance to chemotherapeutic agents and its increased toxicity has given researchers a great challenge to formulate such nanomedicines which are cost-effective, eco-friendly, bio- degradable and most importantly biocompatible. So, natural materials present such benefits. In this era, biosynthesized silk-based nanoparticles could revolutionize the nanopar- ticles in the various biomedical applications most import- antly in the cancer therapy and as a vehicle for drug delivery. The silk-based nanoparticles in cancer therapy is extensively being used and researched because of its less tox- icity, increased biodegradability and biocompatibility. The main benefit is the low cost of silk obtained easily from the silkworm cocoons and could overall decrease the production cost on greater industrial scale. Therefore, if all the results are compiled, this comprehensive review article illustrates the several biomedical applications particularly drug delivery and cancer therapy of the silk-based nanoparticles. However, in near future various effects such as biosafety and long-term cytotoxicity study, immunogenicity, detailed mechanism, and pharmacodynamics researches should be extensively examined in the animal models of the silk-based nanoparticles before taking them to the clinical trials.

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14 M. ELAHI ET AL.

📖 中文全文 Chinese Full Text

中文

# 丝胶与丝素蛋白纳米颗粒——用于癌症治疗的天然产物:综述

Mehreen Elahi, Shaukat Ali, Hafiz Muhammad Tahir, Rabia Mushtaq & Muhammad Farooq Bhatti

**摘要**

蚕丝是蚕的天然化合物,含有两种蛋白质:丝胶和丝素蛋白。这些蛋白质可与其他化合物偶联,形成丝源性纳米颗粒。丝基纳米颗粒在药物递送和癌症治疗中的生物医学应用日益受到关注,因其具有生物相容性、生物降解性、增强渗透性和滞留效应以及较低毒性。然而,目前尚无一篇综述文献专门描述丝源性纳米颗粒的抗癌潜力。在本综述中,我们阐述了:(i)丝素蛋白和丝胶基纳米颗粒的综合概述;(ii)伴随诊断、成像和药物递送等生物医学应用的抗癌机制。

**关键词:** 生物相容性;癌症治疗;生物降解性;药物递送;丝素蛋白;丝胶;丝基纳米颗粒

---

## 1. 引言

尽管在癌症进展和新药研发及各种治疗策略方面已取得巨大进步,但癌症仍然是全球第二大死亡原因。癌症的年发病率约为每年260万例[1,2]。因此,迫切需要采取全球性措施,使工业化地区新型治疗的优势惠及发展中地区,并采取适当行动使现有的癌症治疗手段在欠发达和发展中地区得以推广[3]。最新研究在识别各种癌症的潜在机制和特定病因方面已取得重大进展。目前已有多种癌症治疗方法被应用,包括光动力疗法、放射疗法、化疗、疫苗接种、干细胞移植以及这些方法的联合应用。然而,大多数传统治疗方法费用高昂且会引起有害的不良反应[4,5]。化疗尤其对癌细胞和正常细胞均具有高毒性,从而限制了其临床应用[6]。此外,癌细胞对这些化疗手段产生耐药性是另一个重要问题[7],因此寻找具有生物相容性、生物降解性和低细胞毒性的抗肿瘤药物变得尤为重要。当药物的释放曲线既明确又可控时,药物才能发挥最佳效果,因此需要一种兼具相容性和可调适性的递送系统,例如丝蛋白递送系统[8]。因此,应研究开发经济可行且高效的治疗方案,既能靶向特定癌症,又不会对正常健康组织造成任何损害[9]。

药物递送系统包含载体,活性药物成分以分散、溶解或包封的形式负载于其上,功能组分则连接或吸附于载体上[10]。药物载体分子在药物递送中至关重要,可以微胶囊、纳米颗粒、药丸、微球、乳剂等多种药物释放系统形式给药。其中,纳米颗粒因其作为有效药物载体以提高药物效能的能力而吸引了大量研究关注[11]。这些纳米颗粒还为克服传统药物递送方法的局限性提供了新的可能性[12]。如今,利用纳米颗粒的新型递送系统备受关注,因其有助于将小分子生物活性物质和药物靶向递送至特定部位[13]。

近年来,蛋白基纳米载体因其生物降解性、生物相容性、高营养价值和低细胞毒性而日益受到重视。这些蛋白衍生的纳米载体还表现出高结合亲和力,使细胞能够显著摄取[14]。高效药物递送系统的设计和配制是生物医学领域的核心[12]。纳米载体还因其增强的EPR效应、血液循环时间和主动细胞摄取等特性,提供了更高的抗肿瘤效率并显示最小的副作用[15,16]。生物聚合物涵盖范围广泛,从肝素等生物活性聚合物到壳聚糖、右旋糖酐和海藻酸盐等高分子量药物载体,以及多种多功能分子[17,18]。其中一种天然存在的生物聚合物是蚕丝,已被用于缝合,并获美国食品药品监督管理局(FDA)批准用于人体承重应用。然而,目前蚕丝正作为一种重要的药物递送生物聚合物而兴起[19]。

蚕丝最初含有两种关键蛋白质:丝素蛋白和丝胶[20,21]。蚕丝主要由家蚕(*Bombyx mori*)产生。丝胶成分是蚕茧的可溶性部分,包裹并保持两根丝素蛋白丝在蚕丝茧的丝线中结合在一起[22]。丝素蛋白成分是不溶性蛋白质,具有大量由反向平行β-折叠片组成的疏水结构域[23]。因此,对生物降解性和生物相容性材料的需求表明丝蛋白组分在广阔的生物医学领域中的研究兴趣日益增长[22]。丝胶蛋白因其pH响应性和反应性可用于药物递送应用,这有助于与其他分子结合,从而能够产生小分子[24]。丝素蛋白成分还具有与多种药物结合的高结合能力、样品制备要求和可控的药物释放特性。蚕丝的两种组分均具有优异的生物降解性、生物相容性和极低或无免疫原性[11]。

目前,纳米技术可在药物递送、诊断和癌症治疗中发挥重要作用。鉴于缺乏关于丝基纳米颗粒作为癌症治疗(包括药物递送应用)有前景的综述,我们设计了这篇全面的综述文章。我们首次阐述了丝素蛋白和丝胶基纳米颗粒预期的药物递送和抗癌活性,并就将这些纳米颗粒应用于癌症治疗的临床阶段的关键进展进行了创新性讨论(图1)。

## 2. 生物材料

广泛使用的药理抗癌剂对正常细胞和癌组织均产生许多不良影响,因此化疗具有许多严重的副作用[25]。因此,天然产物如生物材料在组织工程、再生医学和药物递送中发挥着重要作用[26]。

## 3. 纳米技术

纳米技术在许多领域发挥着巨大作用,其应用之一是医学领域的纳米医学。这促进了纳米颗粒形式治疗载体的发展。由于纳米颗粒相比块状物质具有增强的体积比表面积,因此赋予了它们令人瞩目的特性,如增强的机械强度[27]。

纳米颗粒药物载体根据应用通过各种途径开发。虽然设计了多种材料,但其共同目标是阐明药物向定向细胞的转移及其增强的生物利用度,并以最小化的副作用促进应答。由于对正常细胞的毒性以及癌细胞在肿瘤部位对药物的保留较少导致的吸收不良,药物向肿瘤的递送受到阻碍[28]。

分化、细胞增殖和组织再生等许多过程取决于细胞与生物材料表面之间的相互作用[29]。

肿瘤微环境包含细胞、血管、细胞外基质和信号分子[30]。癌是最常见的肿瘤类型(约85%)。在大多数情况下,这些肿瘤是非血管化的,不允许肿瘤生长超过2毫米,也不为其供养形成新的血管[31]。血管生成失调以供养肿瘤细胞,因此形成的血管与正常组织不同。目前,纳米载体被有效地用于癌症的靶向治疗,并运输所需的药物组分,使其能够通过血管系统。一些直径约20-300纳米的纳米载体正在被开发用于药物或许多其他治疗性颗粒的运输。这些治疗旨在通过EPR(增强渗透性和滞留)效应选择性地外渗至癌血管系统[32]。

纳米医学与药物递送研讨会(nano-DDSs)药物仍然是克服耐药性、实现有效细胞内递送的一种有前景的方法。同时,早期研究发现,纳米颗粒可被转运至核周区域的内涵体-溶酶体等区室,远超过膜上的P-gp转运蛋白。这种位置优势允许药物在这些内涵体区室内释放以减少外排泵作用,随后溶酶体和肿瘤细胞的微环境中的低pH可提供pH刺激响应性策略[33]。与游离化疗载体相比,nano-DDSs在血液循环期间可通过EPR效应有效地进入肿瘤部位[34-38]。在这些条件下,包裹在纳米载体中的药物可能直接穿过细胞膜并释放至细胞核,从而诱导大多数癌细胞死亡。因此,以这种方式摄取和释放的纳米药物可能具有更有效的抗肿瘤作用[39]。

## 4. 丝素蛋白

丝素蛋白(SF)是一种不溶性蛋白质,具有大量由反向平行β-折叠片组成的疏水结构域,可作为生物材料在不使用丝胶的情况下被轻易分离[23]。它由蜘蛛和蚕产生,包括家蚕(*Bombyx mori*)和野蚕(*Bombyx mandarina*)幼虫,以及其他蛾属,如*Samia*、*Cricula*、*Gonometa*和*Antheraea*,以及许多其他昆虫属。丝素蛋白的一级结构被归类为天然存在的共聚物单元,包含具有重复小侧链氨基酸(如丙氨酸和甘氨酸)的疏水嵌段,主要包含重复序列(Gly-Ser-Gly-Ala-Gly-Ala)n氨基酸。原始状态含有两种关键蛋白质:丝素蛋白和丝胶,其中丝胶是胶状物质,涂覆在丝素蛋白的两根单丝上[20,21]。这种材料因其低炎症或免疫反应特性以及良好的生物学响应特性而极为适用[40]。丝素蛋白基生物材料正在被开发为纤维、颗粒、薄膜和支架[41-44],并应用于神经、水凝胶、骨、皮肤、血管和软骨组织修复[45-48]。逐渐地,SF因其特性和对其加工的新认识(如弹性、机械强度、可控生物降解性和生物相容性)而被应用于生物医学科学的许多其他领域[49]。SF的这些特性对组织工程特别有益[29]。

### 4.1 丝素蛋白纳米颗粒

乳腺癌一直是女性疾病和死亡不断增长的来源。不久的将来,乳腺癌化疗将包含使用具有高细胞靶向能力的生物相容性药物递送载体,如丝素蛋白,以改善这些问题。使用丝素蛋白(SF)纳米颗粒递送细胞毒性药物提供了最佳的包封率、特异性、增强的治疗指数,以及对乳腺癌细胞的最大毒性,同时对附近正常细胞造成最小或无损害。丝素蛋白负载有合适的化疗药物(如卡铂)。丝素蛋白在许多研究中被描述为向靶向癌症细胞精确被动或主动递送药物的非常有价值的工具,从而确认对附近正常细胞的最大破坏和最小损害,这提高了药物疗效并降低了全身毒性[50](图2)。

### 4.2 丝素蛋白修饰壳聚糖纳米颗粒(SF-CSNPs)

丝素蛋白修饰壳聚糖纳米颗粒(SF-CSNPs)因其低毒性、更好的稳定性、温和的制备程序以及提供多种给药途径而作为药物载体获得了更多关注[51]。SF-CSNPs因其对肿瘤细胞表面的EPR效应而适用于癌症治疗的化疗药物递送。SF-CSNPs通过疏水相互作用在缺陷细胞和组织周围积累。此外,SF-CSNPs的许多物理化学参数被发现是有利的,如穿越生物屏障的能力、保护大分子降解以及将复合物递送至靶向位置的能力[52]。

Yang等[29]证实了肝细胞摄取SF-CSNPs的生物学效应。他们研究了参与泛素-蛋白酶体途径的各种蛋白,并验证了SF-CSNPs可能参与肝癌细胞的增殖和持续[29]。

### 4.3 自组装丝素蛋白纳米颗粒

纳米颗粒的独特特性可以克服在生物医学应用中使用微分子作为治疗剂代表的局限性[53]。例如,药物摄取可通过EPR效应得到改善[54],这可以增加药物在含靶癌组织中的沉积,并通过外排泵介导降低药物耐药性。此外,小分子药物的半衰期和溶解度可通过可控释放作用以及纳米颗粒包封来增强。姜黄素是一种天然存在的姜黄衍生物,含有二酚基团,广泛用于癌症的放射增敏和化疗[55-57]以及生物医学的其他领域[58-60],几乎无毒性[60,11],并作为癌症治疗临床检查中的有前景材料。但由于姜黄素在生理环境中的溶解度差,其在肠道内的摄取或吸收通常很少[62]。最近,姜黄素被包封在SF纳米颗粒中,并被导向乳腺癌特异性细胞,显示出最高的包封率、细胞内摄取和可控释放[28]。此外,已发现两种或更多药物同时给药在癌症治疗中也很重要。由于丝素蛋白的生物相容性和生物降解性特征,它被认为是生物医学领域中有前景的制剂[43,63-66]。SF分子作为药物载体在体内和体外具有可控的分解,可通过改变其分子量、大小、交联度和结晶度来控制[42,44]。SF分子的许多特性使其成为优秀的药物载体。其一,其疏水性氨基酸残基(如甘氨酸、酪氨酸和丙氨酸)通过π-π堆积和疏水界面增强姜黄素和5-氟尿嘧啶(5-FU)的包封,从而提高二元药物转运体的负载效率。此外,亲水性氨基酸残基(如谷氨酸、天冬氨酸和丝氨酸)增强了其在水中的水溶性,并导致在水溶液中形成纳米颗粒[45]。其中一项研究制备了SF纳米颗粒,作为二元药物5-FU和姜黄素的有效负载和转运的生物降解性和生物相容性转运体,并展示了优异的体内和体外抗癌活性。已发现用这种纳米药物治疗可增加ROS(活性氧)的量,从而诱导体外癌细胞凋亡。动物研究表明,接种包封药物的纳米颗粒后肿瘤可显著减少。这展示了用负载纳米颗粒的二元药物治疗乳腺癌的未来可能性[46]。

### 4.4 雷公藤甲素和雷公藤红素负载丝素蛋白纳米颗粒

胰腺癌是一种致命疾病,五年生存率仅为5%。尽管有多种治疗选择,但其诊断率极差。因此,采用不同的疗法来对抗这种致命疾病,例如使用药物组合。雷公藤红素(CL)和雷公藤甲素(TPL)是中药中使用的两种主要化合物,具有广泛的生物活性,其中之一是抗癌活性[48,49,51]。由于丝素蛋白是具有多种独特特性的天然聚合物,因此被用作最佳载体材料之一。其中一项研究通过改进的脱溶剂方法制备了CL和TPL负载的SF纳米颗粒(CL-SFNPs和TPL-SFNPs)[47]。TPL被证明是多种癌症(如乳腺癌、胰腺癌和肺癌)中凋亡和细胞周期的有效刺激因子[14,52,67,68]。它可以有效降低胰腺癌细胞的体外活力,并减少体内肿瘤的转移和生长[49,52,69]。

雷公藤红素通过下调趋化因子受体CXCR4的表达来抑制胰腺癌细胞的侵袭[70]。然而,由于它们的水溶性低和严重的毒性,CL和TPL不能在临床上全身使用。因此,有必要开发用于临床的CL和TPL替代制剂。Ding等[71]分别开发和表征了CL和TPL负载的SF纳米颗粒(CL-SFNPs和TPL-SFNPs)。这不仅克服了疏水性的缺点,还使CL和TPL能够基于最佳剂量和给药方式以及EPR效应在癌组织中被动积累。在CL-SFPNs和TPL-SFNPs的配制、表征和优化后进行了评估。通过使用RITC(罗丹明B-异硫氰酸酯)作为荧光探针,通过流式细胞仪和共聚焦显微镜检查了SF纳米颗粒在MIA PANC-1和PaCA-2细胞中的细胞摄取行为。CL-SFNPs与TPL-SFNPs联合治疗的优势通过人胰腺细胞系(如PANC-1和MIA PaCA-2)中的细胞凋亡和抗增殖检测得到证实[47]。

### 4.5 吲哚菁绿包封丝素蛋白纳米颗粒

在过去几十年中,使用光的多种治疗方法因其显著特性(包括生物相容性、最小侵入性、高选择性和光敏剂的有利定位,从而产生极小的不良反应)而成为传统医疗保健方法的有前景的替代方案[72-74]。光热疗法(PTT)是其中之一,它利用光敏剂,如PTT介质,通过在特定波长窗口将吸收的光能转化为热量来发挥治疗作用[75-79]。近年来,近红外(NIR)触发的抗癌热疗因其出色的组织穿透性、最小侵入性和NIR(>750 nm)的高功效而受到显著关注[80,81]。吲哚菁绿(ICG)是美国食品药品监督管理局(FDA)认证的NIR荧光染料,被用于许多生物医学应用。ICG因其显著特性(包括优异的生物相容性和重要的光热转换效率)而在生物医学领域的诊断、成像和治疗中特别重要[75,76,82,83]。但ICG仍存在一些局限性,包括在水性介质中的稳定性差和肠道吸收、体内浓度依赖性积累导致的体内循环不良、易发生光漂白以及缺乏靶向特异性[77,82,84]。为了克服这些问题,ICG通过各种具有增强稳定性和靶向效率的纳米载体进行运输,包括聚合物载体(PCs)、层状双氢氧化物(LDHs)、脂质体和介孔二氧化硅纳米颗粒(MSNs)等[75,76,83,85-87]。SF重链明显较大,含有C端和N端亲水区域以及十二个富含甘氨酸-丙氨酸的高度重复区域,被内部亲水区域包围[88]。该结构中丝素蛋白的N端区域(FibNT)在低于6.0的pH下经历pH敏感构象变化,从无规卷曲转变为β-折叠片,从而导致负载的治疗载体转移[88,89]。

许多研究报道了丝素蛋白纳米颗粒的形成,包括乳化(>6 μm)、聚(乙烯醇)共混物(300 nm-10 μm)、盐浸出(486-1200 nm)、毛细管微点印刷(25-140 nm)、超临界CO2(50-300 nm)[22,90]和有机溶剂沉淀(35-170 nm)。许多研究已观察到SF纳米颗粒捕获和释放药物的能力[12,22,88,89,91-93]。超临界流体(SCF)技术因其经济特性和环境友好性质,在过去一段时间内受到研究人员的极大关注,用于生产聚合物分子和进一步的治疗性生物活性物质[93-95]。为了实现具有理想生物降解和高治疗效率的多功能双触发装置的制备,通过SCF技术配制了ICG包封丝素蛋白(ICG-SF)纳米颗粒。使用该方法形成的这些ICG-SF纳米颗粒提供了优异的光热稳定性、通过pH响应性从SF中释放ICG(特别是在酸性肿瘤微环境中)以及其被808 nm NIR光的显著激活,从而显著提高了PTT效率。体内外光热试验表明,这些ICG-SF纳米颗粒具有在光触发热疗中破坏癌细胞的能力。这些结果共同表明,通过SCF方法形成的ICG-SF纳米颗粒产生了增强的PTT效率,并可能被用作癌症持续治疗递送系统中的有利材料[96]。

## 5. 丝胶

丝胶成分是一种天然存在的亲水性聚合物,主要由蚕蛾科和蚕科的昆虫产生。它由上皮细胞形成,然后收集在第五龄发育幼虫的广义丝腺腔中。然后从丝腺的中段和后段排出[67]。丝胶约占蚕茧干重的15-20%,将蚕丝茧中的丝素蛋白丝粘在一起。蚕丝中的丝胶在蚕丝纤维脱胶过程中获得,通常在纺织工业中作为废物材料被去除。如今,它在组织工程和生物医学领域提供了令人瞩目的应用[14,68]。丝胶蛋白主要含有氨基酸,如甘氨酸(16%)、丝氨酸(40%)、天冬氨酸、谷氨酸、酪氨酸和苏氨酸。它由极性侧链组成,包含氨基、羧基和羟基,允许容易的共聚、交联以及与更多聚合物组合以配制更好的生物可降解物质[69,70,90]。丝胶的物理化学属性主要取决于丝胶分离的方式和蚕的科属,这影响其功能特征,并将丝胶标记为各种生物医学应用中可能的生物相容性材料[22](图3)。

### 5.1 丝胶自组装/泊洛沙姆纳米颗粒

目前,自组装胶束纳米颗粒已在组织工程中有效地用于定向药物递送目的。在Mandal和Kundu[97]的综述文章中,来自热带地区非桑蚕Tasar蚕茧(*Antheraea mylitta*)的丝胶蛋白与泊洛沙姆F-87和F-127以及溶剂混合,以获得胶束形式的自组装纳米结构,具有携带疏水性(抗癌药物紫杉醇)和亲水性(FITC-菊粉)药物的能力。这些单元在体外研究中使用MCF-7乳腺癌细胞快速进入细胞。使用负载紫杉醇的纳米颗粒对乳腺癌细胞进行的体外细胞毒性检查显示,与单独使用紫杉醇药物相比,具有潜在效果。纳米颗粒包封的药物刺激了MCF-7乳腺癌细胞的凋亡。Bcl-2(抗凋亡蛋白)的下调、Bax(促凋亡蛋白)的上调和PARP(调节蛋白)的裂解表明更多药物诱导的细胞凋亡。该研究将丝胶蛋白开发为在泊洛沙姆存在下生产自组装纳米颗粒的替代生物材料,用于向靶向位置有效递送亲水性和疏水性药物。这为天然存在的丝胶蛋白的纳米颗粒形式提供了新的视角,使其在组织工程和生物医学应用中作为药物递送载体发挥有效和高效的作用[97]。

### 5.2 丝胶-PEG纳米颗粒和丝胶-PBLG胶束

有许多材料被用作纳米载体,但近年来天然聚合物被优先选择。在这些天然聚合物中,丝胶获得了极大的关注。由于其多种有价值的特性,丝胶基物质越来越多地应用于生物医学和组织工程[24,41]。目前,已经建立了许多制备丝基纳米颗粒的方法,如丝胶-PEG纳米颗粒、自组装丝胶纳米颗粒或其他类型的丝或丝胶胶束[89,97-106]。随后,丝胶被视为纳米载体领域的新材料。合成多肽因其固有的生物降解性和生物降解产物的生物相容性而被广泛应用于生物医学领域,如药物递送[107]。合成的聚(γ-苄基-L-谷氨酸酯)(PBLG)已获得关注,并被连接到多糖(如透明质酸)的亲水骨架上[108]或连接合成亲水聚合物(如PEG)[109]以开发核壳设计的胶束。在这些两亲性聚合物单元中,PBLG的核作为疏水性药物的储库,并显著增强治疗剂在血液循环中的稳定性。此外,PBLG可降解为氨基酸,如L-谷氨酸[110]。丝胶-PBLG胶束显示出高稳定性和高药物负载能力,有助于延长血液循环时间。这些胶束还具有可靠的生物相容性、负表面电荷和合适的尺寸分布。纳米颗粒可通过网格蛋白介导的内吞作用有效地内化到细胞中。丝胶PBLG-DOX被携带,然后在远离跨膜药物泵的核周溶酶体中积累,随后在低pH微环境中释放DOX。释放的DOX直接进入细胞核,导致DNA损伤和更大的抗癌作用。主要机制仍有待研究,作为这些纳米载体模式的结果。丝胶-PBLG-DOX胶束的给药可实现适当升高的原生药物浓度,增强DOX对肿瘤的化疗效果,从而减少不必要的细胞毒性副作用,包括对脑、肝、心、肺、肾和脾的损害。丝胶-PBLG-DOX胶束在化疗药物递送中是安全且有帮助的,因此提供了一种可能的技术来逆转多药耐药性[39]。

### 5.3 丝胶调控球形磷酸钙纳米颗粒

磷酸钙(CaPs)是生物硬组织中非常重要的无机化合物,以羟基磷灰石形式存在。由于其优异的生物相容性功能,它们已被用作临床研究中的最佳生物材料。使用预混合快速凝固磷酸钙混合物进行骨修复可显著改善移植物特性并缩短手术时间[111-113]。目前,CaP纳米颗粒的生物医学应用已扩展到许多其他领域,包括基因和药物转移以及体内成像,这依赖于纳米技术领域的进展[114-117]。CaP已被用作转染剂多年,其转染效率已通过改变颗粒大小和进行表面修饰得到增强[118-122]。CaP纳米颗粒为药物递送提供了一组独特的载体,因为它对细胞溶酶体中的酸性pH敏感,且免疫原性小。这允许克服药物耐药性问题。这些CaP纳米颗粒可负载多种分子,包括siRNA、pDNA和化疗药物[123]。CaP纳米颗粒的分散及其在体内的降解对于用作体内药物或基因递送非常重要[124]。

CaP纳米颗粒在静脉注射后的被动靶向仍不限制了其在基因或药物递送中的应用。因此,目前迫切需要一种有效且安全的方法来观察CaP纳米颗粒的递送和体内肿瘤靶向[124]。在先前的研究中,发现丝胶可以保持CaP纳米颗粒的无定形相,具有更高的pH敏感性,使其适用于癌症治疗中的药物或基因递送[125-127]。制备了尺寸为80 nm的S-CaP纳米颗粒,并用近红外染料试剂(DiR)标记(S-CaP@DiR),以检查CaP纳米颗粒在体内的递送和降解,DiR发射的NIR荧光提供了一种非侵入性实时体内成像的浅层方式[128],以清楚地检测体内分散、分解或肿瘤靶向,这是S-CaP纳米颗粒在基因或药物递送中应用的主要原因[124]。

### 5.4 多功能丝胶纳米颗粒

纳米颗粒载体增加了药物的负载能力,并可与疏水性或亲水性药物结合。值得注意的是,它们被设计为具有通过纳米颗粒连接配体主动靶向癌细胞的能力,这些配体明确附着在癌细胞中的分子上。天然聚合物在使用纳米颗粒载体材料方面受到极大关注[129]。这些材料的多功能化学结构使其能够用刺激响应性基团和肿瘤靶向配体进行适当功能化,以设计优异的药物递送系统[130]。这些复合物通过化学键在可能的外部或生理刺激(如酶活性[131]、温度[132]或pH[133,134])存在下在肿瘤微环境中裂解释放材料至肿瘤部位。丝胶富含由羧基、氨基和羟基组成的极性侧链,这使其具有增加的化学反应性和无免疫原性[135-138],并具有多种生物活性和与组织和细胞的优异生物相容性[139,140]。因此,已经发现了多种用于生物医学应用的丝胶制剂,包括薄膜[141]、水凝胶[142]、微粒[143]和3D支架[144]。在纳米颗粒载体领域,已经描述了两种自组装丝胶纳米颗粒用于基因和药物递送,包括丝胶-泊洛沙姆纳米颗粒[97]和丝胶-PEG[69],以及一种脱溶剂丝胶纳米颗粒[145]。所有这些所述的丝胶纳米颗粒都缺乏以专门方式靶向肿瘤的能力。因此,多功能丝胶纳米颗粒是癌症治疗中有价值的工具,具有特异性靶向肿瘤和可控释放药物的特点[105]。

由于叶酸受体在几种人类癌细胞中的表达增加[146],Huang等[105]制备了具有主动靶向肿瘤能力的丝胶纳米颗粒,然后通过受体介导的内吞作用被癌细胞有利地内吞。同时,内吞作用将纳米颗粒转运至具有低pH(酸性)的细胞内内涵体-溶酶体区室。他们设计了pH敏感的腙键来连接丝胶和DOX,从而赋予纳米级递送方法可控的药物释放特性。这种共价连接有助于减少药物的可能泄漏。DOX-丝胶偶联物可由于DOX的疏水性和丝胶的亲水性而自组装形成纳米颗粒。这种方法将使DOX在细胞内成功释放,从而可能增强该丝胶纳米颗粒系统的抗肿瘤效果。该计划被成功实施以制备基于叶酸-丝胶-DOX(FA-SND)的纳米颗粒,并可能被潜在地用作癌症治疗中药物转移的载体[105]。

### 5.5 白藜芦醇负载丝胶纳米颗粒

白藜芦醇的化学名称为反式-3,5,4'-三羟基芪,是一种多酚复合物,已被认为具有抗炎[147]、抗癌[148]和抗氧化[149]特性,使其成为药物中令人关注的生物活性化合物。其抗癌潜力已被广泛研究且特别显著,但其实际应用受到其水溶性差、快速降解和光敏性的限制[150,151]。其中一项研究通过改良脱溶剂方法制备了蛋白质纳米载体,使用从废水中回收的蛋白质粉末丝胶。纳米载体制备通过一系列丝胶蛋白和泊洛沙姆稳定剂浓度进行,允许选择最佳条件以制备小尺寸、球形和稳定的纳米颗粒。丝胶蛋白纳米颗粒通过无溶剂沉淀程序与白藜芦醇(RSV)制备,并添加0.5%(w/v)泊洛沙姆表面活性剂。这些RSV负载的SP纳米颗粒有效抑制了Caco-2(结直肠腺癌)细胞的生长,同时对皮肤成纤维细胞显示无细胞毒性,如细胞活力测定所示。

SP纳米颗粒的细胞内化被证明是表面的,并依赖于孵育时间、载体的转染,体外结果代表RSV的持续释放,以及包封后药物的增强表明其在药物和治疗学中的有前景应用。因此,丝胶蛋白纳米颗粒是一种有利的尝试,可能成为药物运输系统的生物纳米载体[152]。

## 6. 结论与未来展望

各种类型癌症的高发病率及其多种治疗局限性(如药物高成本、化疗药物耐药性及其增加的毒性)给研究人员带来了巨大挑战,要求配制经济环保、生物降解且最重要的具有生物相容性的纳米药物。因此,天然材料提供了这些优势。在这个时代,生物合成的丝基纳米颗粒可以在各种生物医学应用中革新纳米颗粒,特别是在癌症治疗和作为药物递送载体方面。丝基纳米颗粒在癌症治疗中被广泛使用和研究的毒性较低、生物降解性和生物相容性增强。主要优势是蚕丝成本低,容易从蚕茧中获得,总体上可以降低更大工业生产规模的生产成本。因此,如果汇总所有结果,这篇全面的综述文章展示了丝基纳米颗粒的多种生物医学应用,特别是药物递送和癌症治疗。然而,在不久的将来,在将丝基纳米颗粒应用于临床试验之前,应在动物模型中广泛检查各种效应,如生物安全性和长期细胞毒性研究、免疫原性、详细机制和药代动力学研究。