Emergence in protein derived nanomedicine as anticancer therapeutics: More than a tour de force

Journal Pre-proof Emergence in protein derived nanomedicine as anticancer therapeutics: More than a tour de force Zhenchang Wang, Kangkang Zhi, Zhongyang Ding, Yi Sun, Shuang Li, Manyuan Li, Kefeng Pu, Jun Zou

PII:

S1044-579X(19)30412-2

DOI:

https://doi.org/10.1016/j.semcancer.2019.11.012

Reference:

YSCBI 1738

To appear in:

Seminars in Cancer Biology

Received Date:

10 July 2019

Revised Date:

14 November 2019

Accepted Date:

30 November 2019

Please cite this article as: Wang Z, Zhi K, Ding Z, Sun Y, Li S, Li M, Pu K, Zou J, Emergence in protein derived nanomedicine as anticancer therapeutics: More than a tour de force, Seminars in Cancer Biology (2020), doi: https://doi.org/10.1016/j.semcancer.2019.11.012

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Emergence in protein derived nanomedicine as anticancer therapeutics: More than a tour de force Zhenchang Wang,1 Kangkang Zhi,2 Zhongyang Ding,3 Yi Sun,4 Shuang Li,5 Manyuan Li,6 Kefeng Pu,7 Jun Zou,8 1

Department of Spleen, Stomach and Liver Diseases, Guangxi International Zhuang Medical

Hospital, Guangxi, Nanning, 530201, China 2

Vascular Surgery, Changzheng Hospital, Second Military Medical University, Shanghai,

3

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200003, China General Surgery, Wuxi Traditional Chinese Medicine Hospital Affiliated to Nanjing

University of Traditional Chinese Medicine, Jiangsu, Nanjing, 214023, China 4

Oncology Department, Guizhou Provincial People's Hospital, Guizhou, Guiyang, 550002,

China

Department of Respiratory and Critical Care Medicine, First Affiliated Hospital of Jiamusi

University, Heilongjiang, Jiamu, 154003, China

Laboratory Department, Jinzhou Maternal and Infant Hospital, Liaoning, Jinzhou, 121000,

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6

China

Suzhou Institute of Nanotechnology and Nano-Bionics, Chinese Academy of Sciences,

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5

Suzhou, Jiangsu, 215123, China 8

Department of OrthopedicsSurgery,The First Affiliated Hospital of Suzhou University,

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Suzhou, Jiangsu, 215006, China

Abstract Cancer has thwarted as a major health problem affecting the global population. With an alarming increase in the patient population suffering from diverse varieties of cancers, the global demographic data predicts sharp escalation in the number of cancer patients. This can be expected to reach 420 million cases by 2025. Among the diverse types of cancers, the most frequently diagnosed cancers are the breast, colorectal, prostate and lung cancer. From years, conventional treatment approaches like surgery, chemotherapy and radiation therapy have been practiced. In the past few years, increasing research on molecular level diagnosis

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and treatment of cancers have significantly changed the realm of cancer treatment. Lately, uses of advanced chemotherapy and immunotherapy like treatments have gained significant progress in the cancer therapy, but these approaches have several limitations on their safety and toxicity. This has generated lot of momentum for the evolution of new drug delivery approaches for the effective delivery of anticancer therapeutics, which may improve the

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pharmacokinetic and pharmacodynamic effect of the drugs along with significant reduction in the side effects. In this regard, the protein-based nano-medicines have gained wider attention

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in the management of cancer. Proteins are organic macromolecules essential, for life and have quite well explored in developing the nano-carriers. Furthermore, it provides passive or

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active tumour cell targeted delivery, by using protein based nanovesicles or virus like structures, antibody drug conjugates, viral particles, etc. Moreover, by utilizing various formulation strategies, both the animal and plant derived proteins can be converted to

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produce self-assembled virus like nano-metric structures with high efficiency in targeting the metastatic cancer cells. Therefore, the present review extensively discusses the applications of protein-based nano-medicine with special emphasis on intracellular delivery/drug targeting

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ability for anticancer drugs.

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Abbreviations:

rDNA=recombinant DNA EPR=enhanced permeation and retention NPs=polymeric nanoparticles BSA=bovine serum albumin HAS=human serum albumin CNS=central nervous system TGF-β1= Transforming growth factor beta 1 MTX= Methotrexate,

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PLGA=poly-L-lactide-co-glycolic acid PTX=paclitaxel EGCG=epigallocatechin gallate DOX=Doxorubicin MHAP=mesoporous hydroxyapatite LA-Col= lactobionic acid conjugated collagen PEG=polyethylene glycol CAS=sodium caseinate HCPT=10-hydroxycamptothecin ZC=zein-casein RES=reticuloendothelial system THPs=tumour homing peptides (THPs) bZIP=basic leucine zipper proteins VEGFR-1= vascular endothelial growth factor-1 A7R= Heptapeptide ATWLPPR NRP-1= neuropilin-1 WGA=wheat germ agglutinin EpCAM=epithelial cell adhesion molecule avb3= Alpha-v beta-3 MMP-2= matrix metalloproteinase-2 THCPPs=tumour homing cell penetrating peptides NSCLC=non-small cell lung FDA=Food and Drug Administration EMA=European Medicines Agency rHG=recombinant human gelatin FITC-BSA= Fluorescein isothiocyanate labelled bovine serum albumin TH=tetra-H2A rSP=recombinant silk protein OVs=oncolytic viruses T-VEC=Imlygic1 ADCs=Antibody Drug Conjugates VLPs=virus like particles IL-2=Interleukin-2 CXCR4= chemokine receptor type 4

Key words: Cancer, Protein based nanocarriers, Tumour resistance, Tumour drug targeting, EPR effect

1. Introduction Proteins are the essential macromolecules in life, which have pivotal role in biological and structural functionalities of human body including the catalysis, precise interaction and signalling with of biological targets. Generally, proteins or short peptides are formed and engineered by recombinant DNA (rDNA) technologies or by chemical synthesis to mimic the functional activities of the biological importance. Proteins are mainly utilized in industry for catalytic development and in human healthcare applications [1,2]. The patients already lack of having endogenous enzymes or hormones in the bodies can be substituted with drug-

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proteins conjugates to provide additional supplemental therapeutic values [3]. Of course, the proteins can be utilized for the delivery of anticancer drugs including the synthetic agents as well as endogenous nucleic acids and their derivatives [4]. The proteins provide good stability and enhanced deliver ability of the therapeutic substances, owing to their nano-scale size, easy cell penetration ability and site-specific targeting ability, whereas the conventional

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formulation does not fulfil the said requirements [3,4]. In order to improve the enhanced permeation and retention (EPR) effect as well as cell penetrability of the vehicle–drug

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complex [3], nanoscale protein therapeutics are highly valuable.

The chemotherapeutic drugs require cell-targeted drug delivery system [5], but unable to

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meet the desired characters due to poor selective targeted action and severe side-effects [6]. In this context, the drug designing scientists can utilize the natural or bio inspired molecules such as natural viruses or virus like particle [7], surface engineered polypeptides and short

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peptides to achieve nanoscale and targeting [8]. Since for over two decades, the emergence of nanotechnology has paved the way for developing innovative nano-medicine for treating diverse human ailments [9,10,11]. To date several nano-carriers have been reported by the

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researchers and many have reached to the clinics after the regulatory approval [8]. Specifically, nano-medicines in drug delivery offers high surface to volume ratio, small

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particle size and surface tenability characteristics, which play an important role in passive as well as active targeting of drugs [12]. When primary aim of drug delivery is to deliver the active drug molecules into the tumour cells by avoid targeting the healthy tissues by modifying the specific size, surface charge and release of drugs [13,14,15], then nanomedicines prove to be the best system for harnessing the maximal therapeutic benefits [16,17]. Nanomedicines are prepared using different nano-materials of natural and synthetic origin [18]. Among the various nanomedicine-based formulations developed till date, polymeric

nanoparticles (NPs) are considered to be the best carrier systems for controlled and targeted drug delivery [19,20]. However, the use of synthetic polymers for preparation of nanocarriers at times encounter high residue of solvents and surfactants, which may restrict their applications for therapeutic purpose. In this regard, natural bio-molecules including proteins have gained wider attention in drug delivery application due to good biocompatibility and biodegradability [21,22]. As evident from the literature, protein-based nanocarriers have several merits in drug delivery, ostensibly owing to the ease of chemical modification and surface decoration ability. Protein-based nanocarriers can be functionalized by anchoring with various targeting ligands and long-circulating chemical moieties [19,20,23,24], which in

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turn enhances circulation period and intracellular uptake of the particles in the tumour mass. Many factors play an important role in the selection of nanomaterials like solubility, stability, biocompatibility and biodegradability, etc. in drug delivery applications [25,26]. The proteinbased Nanomedicines are very promising due to their minimum toxicity and biodegradable nature. These characters are attributed to the amphilicity in nature and encapsulation of the

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hydrophilic and hydrophobic drugs [27,28]. Proteins NPs can be formed from the natural proteins, which are biodegradable, metabolize and easily functionalize by conjugation of

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anticancer drugs and targeting ligands [16,29–31]. The natural proteins are usually obtained from different sources such as animals (bovine and human serum albumin) and plants (zein

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and gliadin) [32]. Chemical modification of the proteins by the hybrid approach has been recently used for preparation of the recombinant proteins for nanocarriers development [32,33]. Most of the literatures mainly focused on the development and characterization of

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protein-based nanocarriers derived from the albumin, casein, gelatin, etc [32,33]. Therefore, the present article exhaustively discusses the various protein-based nanomedicine and their applications in cancer therapeutics.

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2. Proteins based nanomedicines in cancer therapeutics Animal protein is enriched in meat, fish, dairy, eggs and other animal derived tissues. It has

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great advantage with respect to the abundant availability, biocompatible and nontoxic nature [34,35]. To date, animal derived proteins have been widely utilized as protein nanocarriers. In recent year, albumins NPs are widely investigated in anticancer drug delivery [36,37]. The wide applicability of albumin NPs in drug delivery is attributed to its high drug loading capacity for both water soluble and water insoluble drug molecules. Furthermore, the said nanocarriers can be functionalized with different surface modifying agents to overcome various drug delivery challenges in anticancer therapeutics [36,38,39]. Albumin is mainly obtained from egg white (ovalbumin), bovine serum albumin (BSA) and human serum

albumin (HSA) [40]. Albumin functions as transporter of nutrients and other larger molecules to the cells and it is available as the most enriched plasma protein found in the human body. Abraxane is a marketed albumin nanoparticles formulation by Abraxis Life sciences, USA, for effective delivery of paclitaxel, an anticancer agent to the tumour cells with albumin overexpressed receptors [40]. The solubility studies of albumin were maximum at pH 7.4, which makes it an attractive carrier for delivering a wide range of anticancer drugs [41–43]. HSA found as most enriched protein in human body system with the size of 66.4 KDa [44]. Due to non-immunogenicity, it has been exploited as drug delivery vehicles and implemented as HSA-NPs in treatment of various diseases including cancer [44,45]. Recently, albumin has

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been utilized to develop nanoparticles as carrier for loading and delivering of PTX to treat brain tumour. The developed nanoparticles showed 73.4% entrapment efficiency and particle size is in the range of 100-200 nm. Furthermore, the photomicrographs revealed smooth, spherical shape NPs, thus having the advantage for better uptake efficiency. In vivo study revealed that intravenous administration of PTX-NPs exhibited higher percentage of drug

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reached to the mouse brain as compared to pure drug solution [46–48]. It suggests from the above analysis that the PTX-NP exhibits-controlled release activity and indicated the

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potential for NP crossing BBB. In addition, the above-mentioned research is preliminary and must broaden the targeting studies in vivo in animal models that may include

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pharmacokinetic tests to further assess the efficacy of these formulations and validate selected methods. The most common cancer death among women is breast cancer [49,50]. The predominant pathways responsible for the progression of tumour and metastasis are not

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yet clear. The early detection at the beginning and use of chemotherapeutic approaches can enhance the survival rate and quality of life in breast cancer patients. The therapeutic approaches so far available for the treatment include surgery, chemotherapy, radiation

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therapy, hormonal, monoclonal antibody and immunotherapy. These therapies are found to be associated with high treatment cost, limited success due to high chances of recurrence and

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excessive adverse effects [49,51]. There has been a significant progress in chemotherapy for improving its efficacy in breast cancer patients. However, it still causes harm to the normal cells, and produces adverse effect such as hair loss, diarrhoea and vomiting. This requires the need of a novel system for the delivery of chemotherapeutic drugs [52–54]. Moreover, development of novel system leads to produces liposomes, polymeric NPs, and gold NP, etc for effective delivery of anticancer drugs. The marketed products Nab-paclitaxel (Abraxane®), is an albumin conjugated PTX employed for metastatic breast cancer; but possesses serious side effects, i.e., neuropathy. To minimize the side effects, it must target to

particular type of cancer. In this regard, multiple biomarkers have been discovered to target the highly expressed receptors on breast cancer for targeted drug delivery [49]. Literature reports have suggested the promising delivery of doxorubicin (DOX), paclitaxel (PTX) and methotrexate (MTX) using HSA-NPs for the treatment of prostate cancer and colorectal tumours [41]. Some of these protein nanocarriersare into phase 1 clinical trial. Breast, ovary, lungs, kidney, brain and colon cancer are over expressed with folate receptors [55]. For normal growth of cell, Folic acid (vitamin B9) is required [56]. Folic acid (FOL) is attracted towards the folate receptors, which can be easily anchored to the polymers/lipids, making it an attractive novel carrier for selective drug targeting [55]. The anticancer drugs like TGF-β1

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antibody (TGFAb), MTX and other targeting molecules such as folic acid (FOL) can be utilized to produce the conjugated NPs, as depicted in Fig 1 [49]. This HSA conjugates lowers the production of extracellular TGF-β1 (shown in Fig 2) levels by targeting to TGF expressed tumour cells, whereas MTX provides cytotoxic effect towards the tumour cells (as shown in Fig 3). MTX can be conjugated to FOL to HSA or HSA to TGF-β1 antibody

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(TGFAb) very effectively [49]. The conjugation of FOL to HSA results in enhanced intracellular delivery of FOL-HSA-MTX against human MDA-MB-231 breast tumour cells

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as compared to HSA only (as shown in Fig 4), which over expressed FOL receptors [49]. The conjugation of FOL with HSA helps in higher cellular uptake (as shown in Fig 4).

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Furthermore, the prepared conjugate also causes lower cytotoxicity at the ratio of FOL: MTX (4mM: 6mM) as compared to the MTX alone [49]. The finding of the above study suggests that optimizing multifunctional HSA and HSA's tuneable properties improves the

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effectiveness of such drug-charged NPs. There are various natural proteins which are widely investigated in drug delivery due to their good safety and biocompatibility. Milk protein consists of two main groups namely casein and whey protein [57]. Casein is composed of 1-

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casein, 2-casein, b-casein and k-casein. Further, the casein contains proline enriched proteins, which have both hydrophilic and hydrophobic domains [57]. It is inexpensive, nontoxic and

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biodegradable in nature, and shows good dispersibility in the physiologic media [58]. Another part of milk protein is whey protein, which is typically globular protein and has numerous biological functions. Furthermore, bovine milk, β-lactoglobulin and α-lactalbumin are major whey proteins which are exclusively synthesized in the mammary gland [59]. There are number of literatures reports available on the use of such proteins in the preparation of NPs. Ahmad et al reported the development of casein NPs with the use of different cross linkers such as sodium tripolyphosphate [60] and Genipin using various methods of preparations such as emulsification methods, spray drying techniques and oil-in-water

emulsification [59,61]. Flutamide and alfuzosin hydrochloride as anticancer drugs was encapsulated into the NPs, which resulted in the enhancement of antitumor efficacy and apoptotic effects as compared to the free Flutamide and alfuzosin hydrochloride [62]. Literature reports on casein NPs showed promising nature of the nanocarrier for can deliver controlled intravenous delivery of hydrophobic anticancer drugs [62]. In another study, Zhen et al reported the casein NPs preparation using trans glutaminase instead of glutaraldehyde as the chelating agent [57]. The prepared NPs were used to evaluate the bioactivity of casein in different pH ranges and cisplatin was incorporated into the NPs, which showed extraordinary characteristics to penetrate the cell membrane barriers and effectively inhibit the tumour

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growth [57]. The above research has concluded that cisplatin penetrates the tumour more deeply and impacts the cells far from vascular disease and greater antitumor efficacy in H22 tumour bearing mice than free cisplatin. NPs are also well-dispersed and stable in an aqueous solution. Narayanan et al engineered poly-L-lactide-co-glycolic acid (PLGA) with casein to polymer protein hybrid nanocarriers to encapsulate chemically distinct hydrophobic and

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hydrophilic model drugs [63,64]. This hybrid nanocarrier was utilized for loading of PTX and epigallocatechin gallate (EGCG), which revealed high loading potential for PTX and EGCG,

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high stability, integrity and superior anticancer action [63]. Lastly, the author concludes that the clinical validation carried out on breast cancer cell line supported the enhanced PTX

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therapy together with EGCG and this dual combination will provide significant potential for future translation. Of late, advancement made in the research has shown that casein can be utilized for coating on the NPs which improves their drug delivery performance [65]. The

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casein coated iron oxide NPs used for the encapsulation of DOX and Indocyanine green were found to be stable in presence of gastric protease and gradually degraded in the intestinal protease in simulated intestine conditions [65].

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Such NPs therefore have MRI contrast enhancing capacity for in vivo imaging and promising nanoplatforms for oral drug delivery.Collagen is an animal-based protein found in various

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connective tissues and responsible for the structural integrity of tissues. It is biocompatible, biodegradable and nontoxic in nature [66,67]. Elzoghby et al., reported the use of collagen in the preparation of NPs [68,69]. Collagen can be further utilized in combination with gelatin as a nanohybrid carrier by Lam et al [70]. Li et al, [71] reported collagen as a surface modifier of mesoporous hydroxyapatite (MHAP) NPs. The surface of these NPs can be encapped with lactobionic acid conjugated collagen (LA-Col) MHAP-NPs [71]. The said NPs act as a redox responsive nano reservoir for efficient targeted delivery of the anticancer therapeutics due to excellent biocompatibility, nontoxicity and noninflammatory properties

[71]. Overall, results showed that, under reduced conditions nanocomposites displayed rapid response and burst drug release, and this nano system is a novel stimulus sensitive nano reservoir for liver cancer clinical therapy. Another animal protein is gelatin, which is obtained from acid or base catalytic hydrolysis of collagen [72,73]. It is a macromolecule containing both cationic and anionic moieties along with the hydrophobic groups. In the structure of gelatin, easy chemical modification and covalent drug conjugation can be established. These features are greatly utilized in the making of nanocarriers [72]. Nityananda et al. [72] and Rehana et al. [74] reported the gelatin NPs and its preparation using two step procedures such as desolvation, simple coacervation, solvent evaporation, microemulsion,

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nano-precipitations and self-assemble systems. The gelatin NPs offers several advantages such as high drug loading capacity, entrapment efficiency, better drug release and easy surface modification [74]. Although many key features such as circulating drug stability have been achieved and the emphasis must be on different biological interactions and NP engineering. Moran et al developed the gelatin NPs and DNA was incorporated into it

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[75,76]. Further, the evaluation results of the NPs exhibited superior characteristics such as enhanced intracellular anticancer drug release systems [75]. In summary, the results showed

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that these NPs have excellent properties, highly potent intra-cellular and nontoxic delivery systems, and made them attractive DNA vehicles that can be used for non-viral gene delivery.

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PEGylation is a process, in which polyethylene glycol (PEG) chains are conjugated to proteins and peptides. PEGylation helps in protecting the therapeutic proteins from proteolytic enzymes and its subsequent degradation. Furthermore, it also improves the

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pharmacokinetic drug absorption profile of the therapeutic proteins and causes prolongation of the blood circulation time which results in effective passive drug targeting and lowers the uptake of NPs into the reticuloendothelial system [77]. PEGylated NPs have been studied as

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drug delivery nanocarriers in anticancer therapeutics. PEGylation on gelatin NPs resulted in a two-fold increase in the plasma level as compared to the gelatin NPs without PEG [78,79].

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Over 6-fold increase in the accumulation of NPs in tumour was observed over nonPEGylated NPs [78,80]. Similarly, DOX-loaded PEGylated NPs shows more efficient inhibition of the tumour growth than free-DOX or DOX encapsulated non-PEGylated NPs [81,82]. Ultimately, it is suggested that the NPs are a viable treatment for cancer due to superior antitumor and antimetastatic activity, with low systemic toxicity. Silk fibre (SF) is natural biopolymer which composed of fibroin, which is obtained from the insects’ larvae to form cocoons. Silk fibroin gained wider attention in the drug delivery due to excellent biocompatibility,

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biodegradability

and

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toxicity/immunogenicity

characteristics [83]. Seib et al., reported the use of silk NPs prepared using Bombyx mori and recombinant silks as an anticancer nanomedicine [84]. In another study, curcumin and DOX were encapsulated into silk protein NPs and characterized for treating diseases like cancer and neuroblastoma [84–86]. The combined approaches therefore represented a new class of nano systems that delivered the combinational benefits of the two said drugs. Mishra et al., used silk fibroin (SF) NPs and curcumin as model anticancer drugs encapsulated in it [87]. The SF curcumin loaded NPs were further characterized for various critical attributes like morphology, particle size, encapsulation loading efficiency and better in vitro drug release profile. Therefore, the above-noted NPs were concluded by having biodegradable and

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biocompatible nontoxic systemic delivery of curcumin as a better alternative for long-term drug treatment of tumours [88–90]. John et al., developed silk NPs and DOX to encapsulate into it and found ∼100 nm particle size [91]. Whereas the encapsulated studies of MCF-7 human breast cancer cells find subsequent release of drugs in conjunction with low pH and enzyme degradation. Further, the lysosomal action releases the said drug from silica NPs

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giving a direct evidence for lysosomotropic drug delivery into the tumour cells [91]. Overall the results found that both pH and lysosomal enzymes have an significance in their release

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and have provided the first experimental evidence for the delivery of the lysosomotropic drug [92]. Another drug delivery application of silk NPs in the treatment of neuroblastoma was

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reported by the Coburn et al [92]. The said NPs were utilized for loading of vincristine, DOX and the combinations to give intratumorally sustained release effect in the tumour environment [92]. The findings suggest that intratumoural chemotherapeutic delivery of that

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drug could be a therapeutic technique for pediatric neuroblastoma and could theoretically be applied to other focal tumour types. Similar to other protein-based NPs, the surface modification of silk NPs enhances the properties of NPs as reported by Doudou et al [93]. As

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for the development of surface charge, reversal silk sericin-based NPs are formed by mixing chitosan into silk sericin (SSC). DOX encapsulated in the said NPs provide sustained in vitro

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release of the drug at various pH [93]. Furthermore, it can be concluded that stable SSC@NPs has negative surface charge under mild acidic conditions [93]. The surface charge of NPs undergoes negative to positive conversion under certain pH changes [93]. At the tumour microenvironment, positive charge containing SSC@NPs are triggered and resulted to enhance six-fold higher cellular uptake than the negatively charged SSC@NPs at the pH of 7.4 [93]. Overall, it follows from the above analysis that SSC@NPs was manufactured using a simple method and released the drug in a pH-dependent manner. Another study was conducted by Wang et al., to develop SF and sericin (SS), self-assembled into nanospheres

and nanofibers for delivery of the anticancer drugs [94]. Addition of DOX into the nanospheres and nanofibers, showed marked improvement in the anticancer drug therapy as compared to the free DOX [94]. Our findings have therefore suggested that the selfassembled nanosphere and nanofibers are potentially effective drug carriers for cancer. Another study reported the use of silk with magnetic NPs along with curcumin in the size range of 90-350 nm, which achieved sustained release pattern of curcumin delivery into the breast cancer cells and leading to provide efficient cellular internalization with the help of an external magnetic field for cancer drug targeting [95]. Overall, such NPs are higher for breast cancer treatment and cell internalization is required for the size range, while the magnetic

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core of the NPs has provided a targeted cancer through external magnet. Similar to animal protein, plant proteins such as zein, soy protein, etc. have gained immense popularity in drug delivery area due to their minimum toxicity, easy availability, surface modification possibility with the unbound functional groups and excellent chemical stability. The plant protein is an essential macromolecule in food products [96]. It emerged as potential candidate

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for transportation of anticancer drugs due to unique features like high drug loading capacity and protection of payloads in the NPs [96]. As compared to the animal proteins, the plant

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protein contains higher contents of hydrophobic amino acids and therefore it become more hydrophobic. Plant proteins have the advantage of providing protection from the risk of

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transmission of zoonotic disease, but it is observed more common in case of animal proteins [97]. Gliadin is a natural protein, which is obtained from wheat and other grass genus Triticum but at the same time it has limitations of hydrophobicity, [97]. In the current

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research, Gulfam et al reported gliadin NPs for targeted drug delivery application in various therapeutic domains [98]. Cyclophosphamide is an anticancer drug, which upon encapsulating into the gliadin-gelatin composites NPs helps in controlled release delivery of

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cyclophosphamide and better impact on the tumour killing over use of gliadin alone [98]. In addition, apoptotic activity on the cell lines of breast cancer was shown in cyclophosphamide

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loaded gliadin NPs, supported by Western Blotting study. Therefore, it finally concluded that the NPs can be a powerful tool for the delivery of loaded drugs. Zein is obtained from the maize and it is a water insoluble protein. Luo et al., reported the preparation of hydrophobic zein protein NPs [99]. It is developed by liquid-liquid phase separation method. The said NPs are stabilized by sodium caseinate (CAS) in different composition of zein/CAS mass ratios [100,101]. Lastly, it concluded that zein-CAS NPs cell uptake showed energy-dependent endocytosis cycle and showed potential candidates for cancer therapy. Other researchers have also reported that zein and sodium carboxymethyl cellulose (CMC) combination for

developing zein-CMC NPs [102]. The PTX was loaded into these NPs showed 159 nm particle size, better drug release characteristics and significant tumour inhibition properties [102]. In addition, the cellular uptake and cytotoxicity of the PTX resistant MCF-7 and HEPG2 PTX encapsulated cells of zine CMC was found to be outstanding than free PTX in vitro viability studies. At the same time, flow cytometry experiments have also shown that these NPs are inducing apoptosis to cell death. The latter suggests that the abovementioned NPs displayed significant cancer inhibiting ability.10-hydroxycamptothecin (HCPT) is an anticancer drug which was encapsulated into the zein with FA conjugated targeted NPs and found sustained release of drug with diffusion mechanism. In addition, in vitro cytotoxicity in

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FR-positive Hela cells was increased, and targeted action to folate receptors was improved as compared to free use of HCPT [103]. The aforementioned NPs are therefore an attractive candidate for HCPT encapsulation with sustainable and target delivery. Gahtani et al., reported the use of core-shell NPs prepared using the combinations of zein-casein (ZC), zeinlactoferrin (ZLF) and zein-PEG micelles (ZPEG) [104], which revealed better sustained in

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vitro drug release characteristics after oral drug delivery for long duration of time. Besides, ZPEG micelles resulted in higher cellular uptake, permeability and P-GP efflux inhibition

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over other NPs [104]. ZLF-NPs were readily easily taken up by the intestinal cell which expresses lactoferrin receptors. These three formulations have good mucoadhesive properties

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in the GIT of rats, whereas ZC-NPs were found to possess longest retention in the GIT of rats [104]. Ultimately, the findings show the potential of food protein-based nanocarrier core shell as a source of oral drugs delivery for anti-cancer medicines. In a simpler approach, peptides

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and proteins showed several advantages over the antibodies or virus like particles as drug targeting moieties due to their nano size structure, which exhibit better tumour penetration and minimal chances of escape by the reticuloendothelial system (RES) [105]. Amide group

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in the peptide structure are more prone towards degradation along with less immunogenicity than the full-length proteins or viral capsids. However, the issue regarding degradation of

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linking of the peptides with D-isoforms of the amino acids through cyclation and employing retro-inverse techniques and other various modifications in their chemistry [105,106]. For more than a decade, the use of combinatorial phage display, one-bead one-compound libraries and molecular modelling were introduced to new tumour homing peptides (THPs).Tumour HoPe database (http://crdd.osdd.net/raghava/tumorhope/) is available to provide and collect information until 2012. Artificial aptamer-like peptide ligands are referred by the term aptide and it mimics the DNA expressing site of the leucine zipper (bZIP) proteins [105]. Furthermore, designing of THPs is possible by using computational

approach [87,88,107]. Ample numbers of peptide/proteins have been investigated till date under preclinical studies [108]. Several incorporated peptides of varying types of cargobearing therapeutic NPs are targeted to the tumour cells via action on the over expressed receptors on tumour cell surface. Apart from these, presence of tumour microenvironment with their several components such as RGD, iRGD and ATN-161, which ultimately binds to integrins, E-selectin, P-selectin NRP-1 receptor, SP5-52 with unknown receptor types [108] and lymphatic vessels (Lyp-1 and Syp-1 directed to p32 protein) [109–111]. Apart from THPs, a few natural proteins are also used to develop the nanomedicines, which target the tumoral tissues. Moreover, the nanomedicine utilizes proteins/peptides for targeting tumour

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tissues and presently under clinical trials. Transferrin receptors (Tf) are over expressed on the surface of various tumour cells. The use of siRNA loaded liposomal/polymeric NPs conjugated to Tf ligand can specifically deliver siRNA [112,113] to the over expressed Tf receptors on the tumour cells and provides sustained release of drugs. The integrin αvβ6 is a potential biomarker for non-small cell lung cancer (NSCLC) and other carcinomas identified

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in another study. Whereas this peptide is used for the transmission of αvβ6-specific paclitaxel and conjugates, it resulted in increased cytotoxicity and induces apoptosis compared with free

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paclitaxel in the xenograft H2009 model [114,115].

It also suggests that further studies needed for complete uptake of paclitaxel and for a differe

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nt conjugate too. Likewise, the delivery of a peptide, Angiopep-2, was used to deliver the PTX into the brain tumour cells by endocytosis mechanism with the help of LRP-1 LDL receptor [115]. Angiochem Inc. has designed this product and taken into Phase 1 clinical

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trials. Nab coacervation technology is employed to develop albumin-based NPs and to achieved better stability, half-life and safety [39,116]. Nab-paclitaxel (Abraxane) is USFDA and EMA approved albumin stabilized NPs containing PTX, which is effective against breast

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cancer therapy [39,117]. Apart from this, NPs of carboplatin or gemcitabine is also used in combination against treatment of non-small cell lung (NSCLC) and pancreatic cancer cells

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[118]. The prepared NPs can enter into the tumour cells, stabilizes the microtubules and avoids depolymerization and finally produces anticancer action. Moreover, they also act via inhibition of cell motility, mitosis and replication [119]. In fact, it states that overall survival, advancement free survival and superior therapeutic intervention are significantly improved by the above combination. Another example of albumin stabilized NPs are Nab-docetaxel and Nab-rapamycin. Nab docetaxel acts as taxane microtubule inhibitor and presently it is under clinical trials [120]. Nab-rapamycin bounds the rapamycin with albumin NPs, which shows anticancer action by immunosuppressant and antiangiogenic effects via tumour and

endothelial cell uptake mechanism [121]. Currently, it is under phase II clinical trial for evaluating its efficacy against the advanced malignant perivascular epithelioid cell tumours and also under Phase I/II trial against the non-muscle invasive bladder cancer therapy [121]. Overall, it concludes that Nab-rapamycin is well tolerated in a clinical trial with preliminary evidence of response. Another strategy is conjugation of drugs to the polymeric NPs which enhances the solubility, prevents renal clearance and extends the circulation half-life of the drugs in the blood. Extensively, the drug is linked to this biodegradable water-soluble polymer paclitaxel-polyglutamatein order to facilitate the effective release of paclitaxel over PTX alone [122]. Furthermore, the said conjugated products are under I/II phase of clinical

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trial investigation against NSCLC, glioblastoma, ovarian, head and neck cancer. Overall, the above-mentioned conjugation method is convenient and designed for pharmacokinetics and pharmacological effects. Pegaspargase (Oncaspar 1) drafted PEG with the enzyme Lasparaginase is easily cleared by the mononuclear phagocytic system. Unlike normal cells, leukemic cells are deficient of L-Asparagine enzyme responsible for protein synthesis [123].

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Hence, down regulation of the asparaginase results in the inhibition of protein synthesis, thus eventually leading to the death of tumour cells. Furthermore, Oncospar 1a received FDA and

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EMA approval against the treatment of acute lymphoblastic leukaemia [123]. Therefore, the task of controlling long-term toxicity of NPs is still lacking in specific clinical parameters.

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Therefore, this paper summarizes the therapeutic implementation of said therapy in clinical trials and under preclinical development. 2.1.

Recombinant Protein (RP) Nanocarriers

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RP is employed to develop protein-based NPs, which recently have attracted great attention. Sharma et al., utilizes recombinant human serum albumin (rHSA) for the delivery of 5fluorouracil in the treatment of human colon cancer [124]. It has great advantages such as

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prevention of contamination from blood pathogen. The addition of surfactants in rHSA helps in stabilization of the conformational structures and protects it from the aggregation problem.

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Furthermore, it also maintains the protein bioactivity throughout the shelf life of rHSA [124]. In addition, 5-FU-rHSA-NPs improved cytotoxicity and improved the prolonged release of the drug through i.v. administration. While the therapy and pharmacokinetics of these NPs were higher than 5-FU-NPs. Therefore, the aforementioned nanocarrier system may be a potential candidate for anticancer therapy and testing for anticancer drug translation. Recently, researches have reported the use of recombinant human gelatin (rHG) to develop the NPs which is an alternative over gelatin-based NPs [77]. Whereas the preparation process for the preparation of the said nanocarrier is quick, secure and reproducible. The rHG shows

great potential as protein drug delivery carrier due to its safety. The FITC-BSA encapsulation into the rHG-NPs contribute to drug release without initial explosion and increased protein delivery with reduced toxicity in a biphasic and sustained pattern [75]. This method was therefore first established and showed significant promise with a sustained release profile for protein drug delivery. Moreover, the said NPs were internalized effectively in the tumour cell. Ultimately, the finding indicated that said NPs were appropriate with reduced toxicity for the production of protein drugs. Similarly, recombinant silk protein (rSP) was also developed and was found to possess high thermal stability [77]. Wang et al, demonstrated a new recombinant protein tetra-H2A (TH), which is a product of histone H2A. It is basically a

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protamine alternative and reversible nucleic acid condensing agent [125]. This particular novel protein is utilized to encapsulate the siRNA [125]. Furthermore, TH/siRNA were encapsulated into the inner core membrane layer of lipid based vesicular NPs, which is highly significant in silencing the efficiency of the target gene over the use of protamine conjugated nucleic acid-NPs [126]. The use of that nanocarrier proved biocompatible after repeated

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administration with low immuno-stimulatory and systemic toxicity. The study thus concluded that siRNA will be generated efficiently and will allow the efficient delivery of nuclear acid

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to more of these biopolymers to appear in the near future. Besides, PEG coating on NPs helps in making it a long circulating one and efficient tumour accumulation in the interstitial space

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of tumour tissues. Moreover, after cellular internalization of the NPs get entrapped into endosomes, whereas abundant presence of lipase enzymes in the endosomes to digest the lipid coating and releases core complexes [126]. Besides, in the presence of cathepsin D

releases NA.

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which causes further drop of pH leads to reduced binding affinity with the nucleic acids and

3. Virus-based strategies

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The activity of genetically engineered viruses is majorly based on the uses of different therapeutics such as gene therapy, vaccination and oncolysis [127]. Replicating incompetent

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viruses are utilized in viral gene therapy for transferring the genetic materials to the cancer cells, either to reduce the expression of activated oncogenes to restore the tumour suppressor gene expression or to express the suicide genes. In early clinical trials, current strategies have demonstrated to be safe and effective to produce the desired therapeutic response, which is attributed to the smaller number of tumour cells, which have infected and blocked the immune system [128]. Only Gendicine1 (rAd-p53) for systemic cancer (head and neck carcinoma) therapy has been approved by China in 2003. In combination with the radiotherapy, these therapies provide effective response as compared to the normal treatment

approaches [129]. The use of non-replicating viruses encoding tumour antigens, costimulatory molecules or cytokines, viral-based cancer vaccines are considered as highly effective immunotherapeutic agents [130]. In this regard, adeno virus Instiladrin1 (rADIFN/Syn3) and vaccinia virus PROSTVAC1 are currently under clinical trial of phase III for the treatment of bladder [131] and prostate cancers [132]. PROSTVAC has been investigated in a wide variety of tumour groups with low toxicity with combinative treatment, including radiation, chemotherapy and hormone therapy. Therefore, it suggests that it can happen as both monotherapy and hybrid treatment in future of cancer therapy. Tumour immune evasion and heterogeneous immune responses among patients has hampered the clinical progress of

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virus-based vaccines [133]. The limitations with cancer vaccines to solve the virus-based gene therapy and the oncolytic viruses (OVs) appear to be very useful in controlling the replication of the tumour cells [133]. Further, it induces lysis of cancer cells and also reduces immune-suppression by action on recruiting antigen presenting cells or T cells on the tumour [134]. The efficacy of specific OVs is investigated in preclinical research. The first OV was a

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modified HSV called as Imlygic1 (T-VEC), which is used approved by the USFDA in 2015 for the management of melanoma [135–137]. Ultimately, T-VEC can facilitate the

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immunogenic process of cell death associated with Type 1 interferon signalling, which requires optimum efficacy for various anticancer drugs and further research to resolve the

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intriguing potential. Vaccinia virus Pexa-Vec1 reported in the management of liver cancer, adenovirus CG0070 for bladder cancer, and reovirus Reolysin1 for HNC and retrovirus Toca 5111 for glioblastoma are being evaluated by ongoing Phase III clinical trials

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(www.clinicaltrials.gov). Another example of recombinant oncolytic adenovirus marketed in the commercial name as Oncorine®. It is approved by China Food and Drug Administration (CFDA) and used together with chemotherapy for the treatment of head and neck carcinoma

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[135]. Apart from the potential advantages, OVs as a single agent have limited therapeutic efficacy due to their reduced systemic delivery to the tumour cells and limited maintenance in

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the tumour microenvironment [138,139]. The multimodality approaches combining OVs with radiotherapy, chemotherapy or exhibit promising potential for clinical success [138,139]. 4. Antibody-drug conjugates (ADCs) ADCs are employed for effective delivery of anticancer drugs. ADCs are composed of monoclonal antibody, which covalently bind to an anti-cancer agent [140,141]. The function of ADCs depends on the differential expression of the target antigen in the cancer cells over the normal cells [140,141]. Furthermore, ADCs are employed for loading mitotic microtubule arresting agents such as auristatins or maytansinoids and other cytotoxic agents. DNA

damaging agents including calicheamicins or duocarmycins are under the clinical research. In fact, till date only one product has come in the market and more than 80 ADCs are still under clinical trials [140]. Gemtuzumab ozogamicin (Mylotarg1) is the first ADC, approved by FDA in 2000. Furthermore, calicheamicin antibody drug conjugate is targeted to myeloid antigen CD33 and is effective in acute myeloid leukaemia (AML) patients. Moreover, it has been withdrawn from the market of US and Europe in 2010 due to the toxicity. In 2017, FDA recommended its usage in AML patientsin the low dose [142]. Brentuximab vedotin (Adcetris) was approved in 2011 for the treatment of Hodgkin’s lymphoma and systemic anaplastic large cell lymphoma. It is a conjugated monomethyl auristatin E and anti-CD30

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antibody [143]. Ado-Trastuzumab Emtansine (Kadcyla), has been approved for use in solid neoplasia. It is an anti-HER2 antibody conjugated maytansinoid DM1 used against metastatic breast cancer [144]. Another approved example is Inotuzumabozogamicin (Besponsa1) which is used in acute lymphoblastic leukaemia via targeting the calicheamicin to the CD22 B-cell antigen. Resistance to several ADCs usually happens as with other antitumor agents after

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demonstrating effectiveness in the clinical trials. Resistance occurred due to the changes in the expression of antigen which is identified by the mAb. This mechanism leads to the

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alteration in the ADC internalization and various trafficking pathways, which ultimately disturbs the lysosomal function and drugs starts coming out from the cell via efflux pumps

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[145]. Sixty other products are also being tested in clinical trials. It therefore follows from the above discussion that several ADC resistance mechanisms were used in the clinic and reported techniques to resolve ADC resistance. Therefore, ADCs' potential clinical

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development would benefit from the discovery of resistance drug mechanisms to optimize their therapeutic effect. Currently, researchers have developed a new mAb fragment, which mainly targets the HER2 and/or prolactin [146]. In this analysis, prolactin receptor identified

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throughout breast cancer was linked to the trafficking of HER2 which is the target of clinically accepted ADC ado-trastuzumab emtansine with another target site. In contrast to

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HER2, the prolactin receptor is internalized quickly and constitutively and traffics efficiently to lysosomes. The prolactin receptor is important for encouraging accelerated internalization and triggering HER2 degradation when transferred to HER2. According to this result, low cell surface prolactin receptor concentrations are adequate to mediate successful killing by said receptor ADC, while HER2 ADC needs higher cell surface concentrations of HER2.Therefore, from the above study it is concluded that intracellular trafficking of ADC targets is the key factor for their activity, and further improves their connection to an ADC target, which enhances the internalization of protein.

In addition, this research opens up the possibility of targeting constitutively internalizing high turnover proteins with ADCs and exploited of high turnover protein which has to improve the efficacy of ADCs. Blocking immune screening points was one of the most effective ways to improve the efficacy of anti-tumour activity in patients with cancer. The current clinical report indicates that patients who best react to blockage checkpoint therapy are showing higher CD (+) T cells in the tumour before therapy. The combination therapy of immune control point inhibitors with anticancer drug that increases the number of tumours infiltrating CD8(+) T cells may therefore increase the therapeutic benefit of immune-oncology drugs. Further, currently the adding of ADCs with immune-therapies includes PD-L1 and PD-1

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inhibitors, which expected to show synergistic anticancer action [147,148]. Additionally, ADC research continues to investigate the increased usefulness of various mechanisms for measuring these payloads in various immune cells and to inform rational combinations for clinical development. In this regard, more work is required. In the near future, the use of ADCs using payload explicitly designed to improve immunological responses to tumours

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may be further explored. 5. Virus-like particles (VLPs)

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Bacteriophage viruses such as MS2 particles (VLPs) have various characteristics and are targ eted to deliver therapeutic and imaging agents.Drug delivery systems have adapted VLPs

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usually for vaccine delivery [149]. Furthermore, it can be made more specific by adding targeting protein domain to the upper surface of the viral capsid protein by recombinant DNA technology for production [150]. Various approaches have been employed like

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bioconjugation, electrostatic, physical entrapment etc., which can lead to drug encapsulation within VLPs [151]. Encapsulation of different molecules within VLPs has been used for anticancer targeted therapy. In the same framework, MS2 phase coat protein VLPs expressed

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miRNA or siRNA delivery system based on TAT or Tf that was active against human hepatocellular

carcinoma

cells

[152].

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However, the said VLPs effectively penetrate the cytomembrane and provide miRNA, resulti ng in inhibitory effects on animal models linked to Hep3B, HepG2, and Huh7 cells and Hep3 B.The MS2 Phage VLPs have also been used in another study to encapsulate ricin toxin (Achain) to show the SP94 directed at péptide and the rich histidine fusogen (H5WYG) peptide that induces caspase 3 apoptosis in hepatocellular carcinoma [153]. Alternatively, MS2 VLPs are also used to encapsulate siRNA and have shown higher levels of encapsulated cargo to HCC cell cytosol. In contrast, HCC was found to be ten times higher than endothelic, monocyte and lymphocyte avidity. Based on their multivalent peptide display tolerances and

their ability to encapsule different anti-cancer drugs, selective cytotoxicity of cancer were shown and a characteristic of VLP-based carriers was significantly improved. In addition, the said nanocarrier can both act as cell-specific targeting ligands recognition and as a drug carrirer. Furthermore, advancement in the VLPs forms new phases derived VLPs for active delivery of enzymes. For instance, the successful delivery of encapsulated Cytochrome P450 in P22 VLPs was delivered and targeted to the human cervix and breast carcinoma cells by acting on folate receptors [154]. The majority of the anti-carcinogenic medicines are transformed and catalysed by P450 enzymes. This work includes CYP activity for bacteriophage P22 VLPs. It is immunologically inert and functional for recognition of cell

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carcinoma of the human cervix as well as human breast adenocarcinoma cells. While the CYP was encapsulated inside the capsid virus derived from tumour-treated tumour cells of bacteriophages P22 and VLP, it showed increased tamoxifen cytotoxicity and further increased cellular sensitivity to these medicines. In comparison, PEG coated and folic acid functionalized VLP containing nanoparticles with cytochrome P450 are capable of explicitly

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delivering enzyme activity for pro-drug activation. Therefore, the said nanocarrier may be used to treat certain diseases that lack the action of enzymes. In general, more precise ligand

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development is very necessary and work needs to be carried out to ensure highly specific VLP delivery. Lung adenocarcinoma is the most common type of lung cancer that is

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diagnosed, and is treated with combined surgery, chemotherapy or targeted molecular therapy. Many patients are diagnosed with a progressive and metastatic operable condition which represents the need for effective therapies of lung adenocarcinoma. The lung

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adenocarcinoma has shown a strongly over-expressed surfactant protein B (SP-B). Additionally, JC polyomavirus disease can also affect human lung adenocarcinoma cells. To deliver

a

suicide

gene

entitled

as

SP-B promoter-driven thymidine

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kinase suicide gene (pSPB-tk) to human lung adenocarcinoma JC polyoma virus, VLPs have been explored [155]. In addition, human lung carcinoma and non-lung cells have been tested

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on A549, CH27, and H460. The VLP in combination with ganciclovir (GCV) increased the selective anti-cancer action in the mouse xenograft model. However, the said VLPs were inserted through the tail vein of the mice which inhibits lung adenocarcinoma nodules development by 80 percent. The hybrid strategy can therefore be used as a possible treatment for human lung adenocarcinoma. The development of VLP is a growing field with strong potential in nanoparticles development. The use of empty Johnson grass chlorotic stripe mosaic virus, a member of the genus Aureusvirus, family Tombusviridae, as VLP NPs for drug encapsulation, is used in this field. This nanocarrier was used to load DOX and was

delivered particularly to colonic cell lines [156]. Lastly, it concludes that it was the first work method which approached the said virus for biomedical application and provided a cheap and safe medium for the processing of relatively higher amounts of NPs and could be used as a new nanocarrier to effectively upload and deliver anticancer drug in the future. Another example is Rous sarcoma VLPs, which is obtained from silk worm larvae. While the hcc49 single chain fragment factor was shown. In addition, electroporation introduced the DOX into hcc49 RS-VLPs, and cytotoxicity assays were performed on LS174 T cells. Ultimately, the results showed that the said VLP NPs found significant cytotoxicity on colon carcinoma cells (LS174 T cells) [156]. We may conclude from this study that a nano carrier system can be

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used to provide the target cells with other drugs and gene delivery as anticancer. The study showed that Adenovirus dodecahedron (Ad), which is a biodegradable VLP nanoparticulate protein, is used as a carrier for the delivery of an oncogene blocker in the orthotopic HCC rat model. In this regard co-delivery of the anticancer drug have appeared as novel strategy in the cancer management, i.e., the delivery of DOX with conjugated small anionic nucleotide

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adenovirus dodecahedron VLPs, providing potent synergic effect over human HCC [157]. Whereas in the animal tumour HCC model, Ad-DOX combination induced 40 percent

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tumour growth inhibition and two pro-oncogenes called eIF4E and c-myc were significantly reduced in rat tumour. Therefore, the use of Ad-VLP has improved the delivery of DOX

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which results in statistically significant inhibition of the growth of HCC tumours. The example of hepatitis B core hepatitis protein VLPs has been attracted in the drug delivery because of its favourable structural stability, high uptake capacity, low immunogenicity and

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biomedical biocompatibility. Previously, few attempts were made on cancer therapy. The tumour targeting RGD-HBc is first reportedly manufactured by genetic engineering and the green indocyanine (ICG) was uploaded in the recent studies. The body persistence, aqueous

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stability and target specificity for ICG are significantly enhanced by RGD-HBc VLP [158]. The result summarizes the more accurate and efficient imaging and destruction of the tumour

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U87MG relative to the free ICG. In particular the findings were substantial. The result also indicates that this is a key step in recognizing the functional properties of the HBc VLP and exploring it.

6. Viral mimetics The short peptides have great functional utility in cancer treatment and nanoscale oligomeric assembly of viral capsids have been desirable in the designing of new anticancer drugs and vehicles. Using a recently generated simple protein engineering platform, de novo designed oligomeric protein constructs mimics the character of viral capsids [159]. Empowerment of

these constructs formed by the genetic fusion with peptide ligands greatly helpful in targeting the tumour markers such as CD44 or CXCR4 [160]. Dramatically, the multivalent presentation of these ligands favours the penetration of the cell along with enhancement of effectiveness with the linked drug by conjugation [161]. Furthermore, it achieves significant potential against metastatic colorectal cancer [162]. Colorectal cancer (CRC) cells overexpressing CXCR4 display trafficking functions. Recent research is developing nanoconjugate antimetastatic activity, T22-GFP-H6-FdU, which selectively delivers floxuridine to cancer cells of CXCR4+, inducing DNA damage and apoptosis. Thus, repeated T22-GFP-H6-FdU administration in patients was derived from the CRC models which

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inhibited the 38-83 percent metastasization of mice and showed a reduction of the foci number and size in liver, peritoneal or lung metastasis in mice compared with a reduction of free oligo-FdU based on site expression. The results support the use of this nanomedicine in non-metastatic High-Risk patients for an early metastasis control. This focused approach to drug delivery thus induces powerful antimetastatic effects by metastatic depletion of cancer

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cells CXCR4 + and validates metastatic stem cells as clinical therapy targets. Cancer is managed using synthetic drugs which are systemically administered to achieve effective

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action. The alternative is to use cytotoxic proteins that have been designed to be highly potent drug as effective therapeutic agents such as poisons or venom materials. The new generation

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of anticancer drugs entirely comprised of recombinant proteins which could be market effective in the biopharmaceutical industries for better, more productive and precise cancer treatments, which are expected to lead to growing catalogues of usable venom and toxin

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together with evolving nanobiotechnological goods. As based on the similar principle, many microbial and plant derived toxins are widely explored as the anticancer drugs such as a recombinant version of the plant toxin ricin (the modular protein T22-mRTA-H6) [163,164].

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Moreover, it can self-assemble with CXCR4-targeted nanoparticles can be administered by injection helps in self-targeted delivery of the drugs. In animal models, T22-mRNA-H6

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nanoparticles have shown a remarkable and highly selective therapeutic effect that decreases leukaemia cells. In addition, the said NPs have shown to be nano structures focused on the self-mediated intracellular transmission of toxin for specific oncotherapy. The vehicle-free nanoscale drugs are emerging as novel concept, where external nanoscale vehicle is not required. Highly potent nanostructured diphtheria toxins in colorectal cancer and ricin derivatives in acute myeloid leukaemia have been proven to be highly effective [165]. Therefore, it follows from the above study that toxic protein can be combined with highly selective tumour targeting agent within the nanocarrier can provide the emerging concept of

self-assembled, self-targeted vehicle-free recombinant drugs for accurate medicine. Furthermore, all the above protein derived nanocarriers have been illustrated in Fig 5. 7.0.Conclusion Nanotechnology gives a new platform in the area of biomedical applications with various drug delivery advantages for the cancer treatment. Among nano-medicines, the protein-based nanocarriers have great importance due to their nontoxic, biodegradable and easy surface functionalization ability. There are animal and plant-derived proteins and recombinant proteins that are used to develop protein-based nanomedicines, which have been discussed extensively. Further, combination of functional peptides and supramolecular assemblies gives

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to smart protein based nanocarriers in the form of viral mimetics. The protein-based nanomedicine has been extensively described for anticancer therapeutics via drug delivery, passive drug targeting and specific drug targeting. Apart from these, recent advancement on proteins as natural and synthetic agents was utilized for delivery of anticancer drugs. Besides, the development of protein nanocarriers in combination with other polymers is also referred

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to as protein hybrid nanocarriers which have greatly shown improved characteristics for

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anticancer drug delivery application by overcoming the challenges.

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Conflict of interest: All authors declare none conflict of interest

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Fig Captions

Figure 1: First part of this figure shows two anticancer drugs TGF-β1 antibody and methotrexate, whereas cancer targeting molecule (Folic acid) are conjugated to HSA. Second part is showed reduce extracellular TGF-

β1 level using TGF-β1 antibody, next step is using of folic acid for targeting and the last part demonstrate the higher cytotoxicity of MTX against MDA-MB-231 cancer cell line (Adopted with permission from Ref no

ur

na

lP

re

-p

ro of

20).

Figure 2: The fig shows TGF-β1 antibody-conjugated HSA, it lowers the extracellular TGF-

Jo

β1 levels in MDA-MB-231 cells by targeting to TGF expressed tumour cells (Adopted with permission from Ref no 20)

ro of -p re

lP

Figure 3: This fig represents the methotrexate conjugated HSA nanoparticles and showed

Jo

ur

na

highest cytotoxicity against MDA-MB-231 cells (Adopted with permission from Ref no 20)

ro of -p

re

Figure 4: First part of the figure represents time dependent uptake of folic acid-conjugated

lP

HSA against MDA-MB-231 cells (that shown in Black diamond and HSA in (open square). Second part indicated the cellular uptake of the FOL-HSA-MTX in MDA-MB-231 cells

Jo

ur

na

(Adopted with permission from Ref no 20).

ro of -p re

Jo

ur

na

lP

Figure 5: This fig illustrated the protein derived nanocarriers in oncotherapy.

ro of

-p

re

lP

na

ur

Jo

oo

f

Table

Table 1: Summary of protein derived nanocarriers in effective management of cancer

carriers

Preparation methods

pr

Chemotherapeutic drug

e-

Protein based nanocarriers

Outcomes

References

Albumin Based

Curcumin

Resveratrol

Jo ur

BSA

HSA

NAB technology

Pr

BSA

Curcumin and DOX

na l

HSA

Paclitaxel and

Self-assembly

DOX

[36]

drug delivery This study found neuroprotective effect

[31]

on hippocampal neuronal HT22 cells Coacervation

It received better antioxidant action

[161]

against several cancer NAB technology

This experimental study received

[162]

synergistic and dual drug delivery and

Curcumin

HSA

It received synergistic action and dual

better anticancer action. Drop-wise

It founds tumour targeted action on Gp60

[20]

mixing and

HSA

BSA

f

It received specific tumor targeted action

lactobionic acid

and prevent from drug resistance against

Paclitaxel

to BSA

Hepatocellular carcinoma (HCC).

Cisplatin

Desolvation

It provides sustained glutathione

e-

pr

and

It founds better antibody conjugation and

desolvation

anticancer drug action.

Homogenization

This study provides better stability and

Curcumin

Desolvation

Tamoxifen

difluorinated analog

[44]

[163]

antioxidant action

Modified

Tamoxifen

Jo ur

HSA

Conjugating

Pr

BSA

DOX

na l

BSA

oo

sonication

tumour targeting

[31]

[164]

enhanced anticancer action. It provides folate drug targeting in cancer management.

[165]

Conjugation of Folic

It provides conjugation to HSA and

and MTX

acid to HSA

utilization of folic acid (FOL) which

f

TGF-β1 antibody (TGFAb)

[43]

oo

HSA

results to improves cytotoxicity of TGF-

pr

β1 antibody (TGFAb) and MTX against

e-

human MDA-MB-231 breast cancer cells over HSA only.

Recombinant Based 5-fluorouracil

Pr

Recombinant human serum

New recombinant protein tetra-

Casein based nanoparticles

contamination. Further it also reported better stability and effective delivery

It found higher encapsulation of siRNA

TH/siRNA

Jo ur

H2A (TH)

[119]

against human colon cancer.

na l

albumin (rHSA)

It found better safety in term of no risk of

[121]

and showed a higher gene silencing efficiency.

Casein Based

Flutamide and alfuzosin

It prepared by

It founds enhanced uptake of said drugs

hydrochloride

emulsification

and apoptotic effects as compared to free

methods, spray drying

[39]

Flutamide and alfuzosin hydrochloride

f

techniques, and oil in

Cisplatin

Emulsification

The said nanoparticles showed

methods, it utilizes

extraordinary characteristics such as

e-

Casein nanoparticles

pr

oo

water emulsification.

effective cell membrane barriers and

the place of

effectively targets and inhibit the tumour

Pr

transglutaminase in

growth.

na l

glutaraldehyde

Emulsification

Casein coated iron oxide

DOX and Indocyanine

Double emulsification

The casein coated iron oxide

nanoparticles

green

methods

nanoparticles have been utilized for

Casein with poly-L-lactide-co-

PTX and epigallocatechin

The said drug loaded LPHNs founds

glycolic acid (PLGA) called as

gallate (EGCG).

methods

higher drug loading, stability, integrity

Jo ur

polymer protein hybrid

[52]

[59]

and enhanced anticancer actions.

nanocarriers (LPHNs)

encapsulated the DOX and Indocyanine green, it founds stable in the gastric pH

[60]

f

and gradually degraded under the

oo

intestinal protease in simulated intestine condition. Furthermore, it revealed

pr

higher cytotoxicity in cancerous cells.

Lactobionic acid

Pr

Collagen as a surface modifier of mesoporous hydroxyapatite

Jo ur

na l

(MHAP) nanoparticles.

Gelatin nanoparticles

e-

Collagen Based

DNA

The said lactobionic acid conjugated

[66]

collagen (LA-Col) nanoparticles acted as redox responsive efficient targeted anticancer therapeutics. Further it gained excellent biocompatibility, non-toxicity and non-inflammatory action.

Gelatin Based Desolvation, simple

The said nanoparticles exhibited

coacervation, solvent

effective intracellular delivery of DNA

evaporation,

and thusmake it as potential non-viral gene delivery systems in cancer therapeutics.

[70]

DOX

Simple coacervation

The DOX loaded said nanoparticles

f

PEGylated Gelatin based

[72]

showed efficient tumour growth

oo

nanoparticles

inhibition over the free DOX or DOX

pr

loaded encapsulated non-PEGylated NPs.

Curcumin and DOX

Solvent Evaporation

DOX

Jo ur

Silk protein-based NPs

na l

Pr

Silk protein-based NPs

e-

Silk protein based

Silk nanoparticle

Vincristine, DOX or the

Solvent Evaporation

nanoparticles (SSNPs)

DOX

[79]

action against various cancer including neuroblastoma.

It founds effective lysosomal escape by

[86]

developing of lysosomotropic action, resulted to better killing of tumour cells. Solvent Evaporation

combinations

Silk sericin based

It founds better therapeutic anticancer

It provides intratumorally sustained

[87]

release system. It formed by mixing

The said NPs undergoes a negative to

of chitosan into silk

positive conversion under tumour

sericin by using of

microenvironment. At this environment,

[88]

the surface charge NPs are triggered and

f

Solvent Evaporation

oo

resulted to enhance six times higher

cellular uptake than the negative charge

pr

containing SSNPs.

Paclitaxel

Liquid-liquid phase

Pr

Zein and sodium

e-

Plant Protein Based

carboxymethyl cellulose

separation

combined zein CMC

na l

nanoparticles.

The said NPs founds betterin vitro drug release characteristics and higher tumour inhibition.

Zein with FA conjugated

10-hydroxycamptothecin

Liquid-liquid phase

The result found better antitumor action

targeted nanoparticles

(HCPT)

separation

as compared to free use of HCPT

Jo ur

[94]

[98]

DOX; Doxorubicin, HCC; Hepatocellular carcinoma; HSA; Human serum albumin, NPs; Nanoparticles, SSNPs; Silk Serin Based Nanoparticles, FA; Folic acid, CMC; Carboxymethyl cellulose, BSA; Bovine serum Albumin, PEG; Polyethylene glycol