《现代食品工程高新技术》课程教学资源（文献资料）食品粉碎、微胶囊包埋新技术 Recent advances in novel drug delivery systems and approaches for management of breast cancer：A comprehensive review.pdf_大学文库

Contents lists available at ScienceDirect Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst Recent advances in novel drug delivery systems and approaches for management of breast cancer: A comprehensive review Umme Hania,∗ , Mohamed Rahamathullaa , Riyaz Ali Osmanib , Honnavalli Yogish Kumarc , Deeparani Urolagind , Mohammad Yousuf Ansarie , Kamal Pandeyf , Keerthana Devig , Sabina Yasminh a Department of Pharmaceutics, College of Pharmacy, King Khalid University, Guraiger, Abha, 62529, Saudi Arabia b Postdoctoral Research Associate, Indian Institute of Technology, Bombay, India c Department of Pharmaceutical Chemistry , JSS College of Pharmacy,JSS Academy of Higher Education and Research, S S Nagar, Mysuru-570015, Karnataka, India d Department of Pharmaceutics, Aditya BIPER, Bangalore, Karnataka, India e Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), Industrial Area, Hajipur, Bihar, 844102, India f Product Development officer, Asian Pharmaceuticals Private Limited, Nepal g Saveetha College of Pharmacy, Chennai, Tamil Nadu, 600128, India h Department of Pharmaceutical Chemistry, College of Pharmacy, King Khalid University, Guraiger, Abha, 62529, Saudi Arabia ARTICLE INFO Keywords: Breast cancer Liposomes Hydrogels Exosomes Dendrimers Microspheres Microbubbles Phytosomes Micelles ABSTRACT Breast Cancer (BC) is a carcinoma of breast tissues, the major recurrent cancer in women and also the foremost cause of death with approximate 5 million annual deaths worldwide. The most developed regions of the world show the highest incidence rate of BC when compared to less developed regions. However, the mortality rate was found to be higher in developing countries. In women with less than 40 years of age, approximately 7% diagnosed as suffering from BC and in women with less than 35 years of age, it was less than 4%. Presently less curative options are available for such patients. Emerging novel delivery systems may result in a promising approach for its early recognition and efficient treatment. Currently, cancer research focuses on improving BC treatment using various novel delivery systems of chemotherapeutic agents such as nanoformulations, liposomes, hydrogels, exosomes, dendrimers, microspheres, microbubbles, phytosomes, micelles, etc. The present review encloses existing assorted novel drug delivery systems and approaches intended for diagnosis and treatment of BC. 1. Introduction The breast cancer (BC) is the most frequent cancer having immense mortality among women worldwide that estimated by the International Agency for Research on Cancer (IARC) as over two million new cases and approximately five million deaths (2014). It is the most lethal cancers in spite of the incredible advancement of diagnosis and treatments [1,2]. In a breast, the terminal duct of lobule unit having epithelial cells lining are main origin of breast cancer (BC). This cancer cells can be classified as non-invasive or in situ (in the same place). This cancer cells are present inside basement membrane of the elements of draining duct and terminal duct of lobular unit. . When the cancer cell spreading occurs exterior to the basement membrane of the lobules and ducts into the neighboring adjacent normal tissues it is called invasive BC [3]. There are different types of BC that include like ductal carcinoma, lobular carcinoma, and in- flammatory mammary cancer [4]. The Tumor, Node, Metastasis (TNM) is the system used to classify various stages of breast carcinoma. The early stages of invasive carcinoma are the TNM stage I, TNM stage II and TNM stage IIIA and most of these tumors are considered operable traditionally. Fig. 1 depicts the various stages of BC. More than 90% of BC diagnoses are made early and can potentially cured by following therapies are surgery, radiation and systemic. In early BC patients receiving appropriate treatment, 5 year survival rates are in excess of 75% [5]. Now a day's less curative options are available for the treatment of the BC patients. Emerging novel delivery systems may result in promising approach for the early recognition and its possible treatment. Currently, cancer research focuses on improving BC treatment using various novel delivery systems of chemotherapeutic agents. These novel drug delivery systems include nanoformulations [6], liposomes [7], https://doi.org/10.1016/j.jddst.2020.101505 Received 12 February 2019; Received in revised form 25 December 2019; Accepted 5 January 2020 ∗ Corresponding author. E-mail addresses: uahmed@kku.edu.sa, ummehaniahmed@gmail.com (U. Hani). Journal of Drug Delivery Science and Technology 56 (2020) 101505 Available online 08 January 2020 1773-2247/ © 2020 Elsevier B.V. All rights reserved. T

hydrogels [8], exosomes [9], dendrimers [10], microspheres [11], microbubbles [12], phytosomes [13], micelles [14], Nosrati H et al. reported the preparation modified iron oxide magnetic nanoparticles (IONPs) functionalized with L-lysine (Lys) and L-phenylalanine (PhA) loaded with curcumin (CUR). They were studies for their effects, loading capacity, the release profile of CUR, biocompatibility and anticancer activity. CUR loaded amino acids modified iron oxide magnetic nanoparticles (F@AAs@CUR NPs). The anticancer activity results of free CUR was found to be more than the activities of F@Lys@CUR NPs and F@PhA@CUR NPs due to the CUR releases pattern from the F@ Lys@CUR NPs and F@PhA@CUR NPs are slower than the diffusion of free CUR [15]. Danafar H et al. established a reliable micellar delivery system using monomethoxypoly (ethylene glycol)–poly (e-caprolactone) (mPEG–PCL). The encapsulation of SF inside mPEG–PCL as a nano-carrier was established and the cytotoxicity assay against human breast cancer cell line was evaluated. Based on the cytotoxicity results of mPEG–PCL micelles against human breast cancer cell line (MCF-7) suggested that SF/mPEG–PCL micelles can be an effective breast cancer treatment strategy in the future [16]._ENREF_15. Hence, most up-todate conclusions and any crucial findings of BC treatment required to be broadly spread to scientific, medical and research societies. Like our previously published article on cervical [17] and prostate [18] cancer in the present review, we have focused on diverse delivery systems existing for BC treatment and an outline has depicted in Fig. 2. 2. Pathophysiology of breast cancer Breast cancer is a proliferative carcinoma type originated from breast tissue [19]. Signs of the BC may include the shape changes, dimpling skin, nipple leakage [20]. Symptoms of spread may include swelling of breast tissue, lump associated with pain and discoloration [21]. Proliferative breast lesions include ductal hyperplasia, sclerosing adenosis, fibroadenomas and intraductal papillomas [22]. In ductal carcinoma, hydroxyl radicles cause lesions and lead to cancer pathogenesis [23]. Epidemiology of BC suggests that stimulation of estrogen in the absence of cyclic progesterone secretion develops breast carcinogen, as the estrogen receptor alpha has a vital role in the normal development of breast cells. Depletion in the chances of BC can be done via the protective action of breastfeeding; which reduces the level of sex hormones responsible for cancer [24]. Structural classification of BC includes, cancer originating in lobules, ducts, skin, and molecular point of view which includes estrogen receptor-negative tumors and enriched human epidermal growth factor receptor (EGFR) 2. There are estrogen receptors positive tumor subtypes viz. luminal A tumor and luminal B tumor [25]. Based on the receptor (progesterone, estrogen) on the cell nucleus and it is over-expression, the type of BC can be identified [26]. Cancer of the breast is very common among women. Factors responsible for cancer are hormonal, environmental, personal and genetic risk factors [22]. Woo et al. have suggested that antimutagenic agents have induced cell death in MCF7 BC cells by EGFR expression reduction. The down-regulation of EGFR was caused by protein destruction [27]. Franco et al., suggests that altered expression of the different gene isoforms can cause carcinogenesis and gene isoforms essential for epithelia generation. In the recent investigation, it is found that altered gene isoform expressions have a significant role in BC development [28]. Cellular morphology of breast cancer is depicted in Fig. 3. 3. Nanotechnology-based novel drug delivery systems Nanotechnology-based therapeutics is highly used in cancer therapy for enhancing drug solubility, stability, and decreasing multidrug resistance as well as to enhance the safety and efficacy of cancer treatment. Nanoparticles, dendrimers, polymeric micelles, liposomes, polymeric drug conjugates, exosomes, and polymersomes are some of the efficient carriers in nanotechnology-based drug delivery systems; which Fig. 1. Different stages of breast cancer with cell cytology. Fig. 2. Diverse delivery systems and approaches are available for breast cancer therapy. U. Hani, et al. Journal of Drug Delivery Science and Technology 56 (2020) 101505 2

are presently investigated extensively for augmented cancer therapy [29]. 3.1. Nanoparticles Nanoparticles (NPs) are rapidly emerging nanocarriers which can be defined as ultradispersed solid supramolecular structures with size ranging between 10 and 1000 nm. Potential interest in the field of nanopharmaceuticals has generated several advancements recently. NPs has a potential impact on the treatment of cancer, in which the drugs can be either, encapsulated, entrapped, dissolved or attached to an NP matrix acting as a reservoir for drug [17,30]. Poly (lactic-co-glycolic acid) [PLGA], polyethylene glycol (PEG) and modified PLGA have commonly used polymers for the preparation of NPs. A diverse work has been carried out by a few researchers to prepare NPs using PLGA and PEG. Star-shaped copolymer (of mannitol core PLGA-TPGS diblock) based NPs loaded with docetaxel were prepared using a modified nanoprecipitation method. The resulting NPs showed a significantly greater level of cytotoxicity; which may be due to increased encapsulation efficiency and drug loading in comparison with commercially available Taxotere® formulation. Moreover, the higher antitumor efficacy of prepared NPs was revealed from in vivo studies [31]. A novel polymeric nanocarrier, Cholic acid-core star-shaped PLGA based NPs loaded with simvastatin were developed to achieve controlled and sustained delivery of drug consequently resulting in a significantly greater level of cytotoxicity. As expected, developed NPs effectively inhibited the growth of tumors for a longer time period by internalizing into MDA-MB-231 human BC cells and dramatically inhibited the expression of the cell cycle protein cyclin D1 when compare to pristine simvastatin and linear PLGA NPs loaded with simvastatin [32]. NPs can also aid in enhancing the solubility of poorly water-soluble drugs such as curcumin. Encapsulation of curcumin in PLGA NPs significantly protects curcumin from the environment and improves its bioavailability and thereby its release in cytoplasm resulting in G2 receptor blocking action on MCF-7 cancer cell lines [33]. PLGA NPs loaded with 1,7- bis [3,4-dimethoxyphenyl]-5-hydroxyhepta-1,4,6-trien-3-one [ASC-J9] were prepared to enhance the bioavailability of ASC-J9; which has been proposed for the treatment of BC. ASC-J9 release causes G2/M blocking effect on the cell cycle leading to inhibition of cellular growth [34]. To increase the lapatinib antitumor activity a novel polymer-lipid hybrid nanosystem having poly (lactide-co-glycolide)-Da-tocopheryl PEG 1000 succinate coated by a lipid layer of PEG was developed. The prepared nanosystem has efficiently induced carcinoma cell apoptosis when compared to free drug. NPs by dropping the uptake of the drug by the reticuloendothelial system (RES), have significantly prolonged its blood circulation time; which ultimately results in drug accumulation in cancer tissues and thereby resulting in a maximum therapeutic effect in the BC treatment [35]. As per another reported study, active triphosphates of fluxuridine, gemcitabine and cytarabine nucleoside analogs encapsulated in PEG-cl-PEI or F127-cl-PEI nano-gels exhibited similar cell cycle perturbation and cytotoxicity in vitro, equal tumor growth-inhibitory activity and faster cell accumulation in vivo at very low dose in comparison with free drug [36]. Hydrophobic drug paclitaxel (PTX) loaded dendrimer stabilized smart NPs prepared to achieve targeted delivery have showed a pHdependent release of a drug. The fabricated NPs were found to be stable at physiological pH, and quick release of drugs in the tumorous environment was noted with efficiently suppressed cancer cells growth and significantly induced apoptosis. These NPs were also reported to be extremely biocompatible than PTX [37]. PTX can also be delivered effectively with bioactive waterborne polyurethane nano-micelles, an effective novel nanocarrier proposed by Khosroushahi et al.; which significantly inhibits human BC MCF-7 cell growth and proliferation, and results in apoptosis due to liberation of cytochrome C in the cells [38]. Silica NPs loaded with dual drugs (PTX and suramin) using triple targeting ligands specific towards neoangiogenesis and cancer, for achieving synergistic targeting, were successfully synthesized by Veeranarayanan et al. Reportedly, elevated uptake of NPs was observed thereby imparting superior therapeutic efficacy against BC cells along with complete immobilization disabling the migration ability of activated endothelial cells [39]. To increase the aqueous solubility of pyropheophorbide, it was entrapped in organically modified folate receptor-targeted silica NPs. It has been observed that the uptake of pyropheophorbide in squamous cell carcinoma was increased with a decline in pH of the incubation media and uptake was not influenced for plain NPs [40]. Phosphorylated drugs can be delivered efficiently in a sustained manner with the aid of nanoformulations. Considering this fact, researchers have fabricated polymeric conjugates of nucleoside analog for the augmented treatment of drug-resistant tumors. It has been observed that the developed system exhibited rapid enzymatic release of floxuridine 5'phosphate resulting in enhanced cytotoxicity. Hence, the fabricated system was claimed to be significantly effective in the management of drug-resistant tumors [41]. Antioxidants such as Vitamin C and E encapsulated NPs and silver NPs were prepared from Hibiscus rosa-sinensis petal extracts and chitosan. The polymer chitosan posses targeting ability and is biocompatible, biodegradable and cationic. Prepared nanoformulations were found to be extremely haemocompatible and showed good encapsulation efficiency of about 76%. Moreover, significantly higher anticancer activity was observed against BC cell lines (MCF-7) [42]. Human monoclonal antibody trastuzumab was employed to target human EGFR-2 positive cancer cells. Trastuzumab guided lipid-based NPs along with rapamycin composition (an imaging agent) were analyzed for targeted BC therapy. The results revealed improved therapeutic efficacy of the drug in NPs formulations compared to pure drug. The developed targeted multifunctional NPs exhibited excellent bioimaging in 2D monolayer and 3D tumor spheroid models and hence claimed for potential implications in better cancer management [43]. Self-assembled chitosan NPs were prepared in another research vocation encapsulating damnacanthal; an upcoming potential candidate for suppressing cyclin D1 expression at the post-translational level in dose and time-dependent manner. The prepared NPs exhibited good yield, drug loading and revealed better inhibition of cell growth when compared to that of non-encapsulated damnacanthal [44]. Fig. 3. Cellular morphology of breast cancer. U. Hani, et al. Journal of Drug Delivery Science and Technology 56 (2020) 101505 3

Gold NPs can be targeted to mitochondria of BC cells and could helpinduce apoptosis and trigger cell death; which can be used in photothermal therapy of BC [45]. Enzyme sensitive amphiphilic peptide dendritic copolymer-based doxorubicin (DOX) NPs exhibits effi- cient apoptosis of cancer cells in vitro by retaining and accumulating within tumor cells for a longer time period and also reduces DOX-induced toxicities. Hence, these can be a prospective drug delivery vehicle for augmented BC therapy [46]. A significantly high antitumor activity can be achieved by an enzyme response peptide dendrimer DOX conjugate based NPs which can induce cell death on the 4T1 breast tumor model [47]. The pH-responsive NPs of dendronized heparin DOX conjugate for cancer therapy had been proven to provide high antiangiogenesis effects, strong antitumor activity and induce apoptosis in BC cells [48]. Physical instability is a common drawback of nanoformulations. In an approach, researchers used D-glucose to stabilize the gelatin/collagen-based matrix wall to deliver the Calendula officinalis powder and oil. NPs loaded with Calendula officinalis powder in comparison with free powder, has significantly improved the anti-cancer activity towards human breast adenocarcinoma MCF7 cells and human hepatoma SKHep1 cells [49]. Free rhodium citrate and rhodium citrate loaded maghemite NPs have proved systemic action in treating BC; which was analyzed by hemogram, alanine aminotransferase, iron and creatinine serum level, and histology of kidney, liver, and lungs [50]. Triple-negative breast cancer (TNBC) is considered as one the most invasive cancer with a yearly increase in a number of incidences; demanding urgent development of effective therapeutic options and strategies. In a research approach particle, size-reducible and angiopep-2 modified NPs were fabricated with the aim of increasing accumulation and penetration deep down in tumor tissue. In this work for enhancing NPs cancertargeting efficiency, angiopep-2 was anchored on NPs surface to smooth the progress of accumulation through binding with TNBC overexpressed low-density lipoprotein-receptor related protein (LRP). Whereas, for attaining high penetration and increased tumor retention, particle size-reducible NPs were fabricated using gelatin NPs (GNP) comprising of dendrigraft poly-lysine (DGL) loaded with doxorubicin (DOX). These NPs were found efficient in tumor targeting as evident by in vitro cellular uptake studies and ex vivo imaging. The larger size NPs were size reduced (from 185.7 nm to 55.6 nm) via degradative action of matrix metalloproteinase-2 (MMP-2). Moreover, NPs ability to penetrate was dominantly improved in tumor spheroids post-incubation with MMP-2. Because of the combinatorial approach of tumor targeting and enhancing penetration; developed NPs were promisingly effective in tumor growth inhibition (with tumor growth inhibition rate of 74.1%) in a mouse model bearing 4T1 BC. In short, efficacy and potential applicability of nanoplatforms based combinatorial approach for the treatment of TNBC were put forth through this study [51]. In another study of the same basis, Ruan et al. have reported a new-fangled multistage system exhibiting targeting effect as well as size changeable properties to inhibit tumor growth and metastasis. The multistage system was comprised of numerous small size gold NPs (AuNPs) fabricated onto the surface of matrix metalloproteinase-2 (MMP-2) degradation prone gelatin nanoparticles (GNPs). Anticancer agent DOX was then tethered on AuNPs through a pH-sensitive hydrazone bond followed by surface decoration using a tandem peptide of arginylglycylaspartic acid (RGD) and octarginine for improving the efficiency of tumor targeting. The fabricated NPs based system (G-AuNPs-DOXRRGD) was reported to release DOX in pH-dependent mode and exhibited shrinking behavior post 24 h incubation with MMP-2 (from 185.9 nm to 71.2 nm). Superior penetration efficiency was confirmed from collagen diffusion and tumor spheroid penetration study results. In vivo evaluation of G-AuNPs-DOX-RRGD in 4T1 xenograft bearing mice revealed active targeting of 4T1 tumor followed by interstitial matrix penetration and deep tumor accumulation. Hence, researchers concluded exceptional anti-tumor potential of developed multistage nanosystems which has been attributed to the adopted synergistic approach [52]. Mesoporous silica nanoparticles (MSNs) are found to be excellent nanotechnology for anticancer drug delivery. They were found to be stable by virtue of their interesting properties for biomedical applications such as high chemical stability, large surface area, and tunable pores diameters and volumes, allowing the incorporation of large amounts of drugs. Sabio RM et al. presented an overview of preparation methods of MSNs and their properties that affect drug delivery [53]. Methotrexate (MTX) with glycine (F-Gly NPs) coated magnetic nanoparticles (MNPs) to get conjugates (F-Gly-MTX NPs) were prepared by an amidation reaction via the co-precipitation method. Biocompatibility was determined by hemolysis assay and cytotoxicity studies on HFF-2 and HEK-293 cell lines. In these MNPs, the drug delivery depends on the release of the MTX by peptide bond cleavage inside the compartment of lysosomes [54]. Magnetic nanoparticles (MNPs) plays a vital role in the identification of cancerous lesions. At cellular levels, MNPs having specific magnetic characteristics and biological interactions. MNPs created a revolution in the diagnosis and clinical treatment of the disease. Arginine functionalized Iron oxide MNPs were conjugated with MTX. Cell cytotoxicity test on the normal cell line (HFF-2) and hemolysis assay confirmed that synthesized nanoparticles were biocompatible. Release study was performed in low pH conditions with and without proteinase K. Here, MTX was released through peptide bond cleavage with proteinase K at acidic pH [55]. Tamoxifen (TMX) loaded L-tyrosine natural amino acids (Tyr) modified Fe3O4 magnetic nanoparticles (F@Tyr@TMX NPs). Tyrosine, which was containing phenol groups was selected to study their effects on biocompatibility, loading capacity and release profile of TMX. It was observed that F@Tyr@TMX NPs exhibited a more cytotoxic effect compared to that of free TMX [56]. A novel dual-target recognition sandwich strategy for selective capture and detection of MCF-7 breast cancer cells based on core-shell magnetic mesoporous silica (Fe3O4@nSiO2@mSiO2@apt) nanoparticles was developed. This assay showed high specificity and sensitivity to the target MCF-7 cells. Therefore, the approach proposed here may have great potential for early breast cancer diagnosis [57]. Asgari M et al. presented their research work on a novel method based on an inverse microemulsion system to synthesize monodisperse magnetic mesoporous silica nanoparticles (MMSN) with core-shell structure. The preparation is basically w/o microemulsion system which is silica precursor containing cyclohexane as a continuous phase and magnetic seeds (Fe3O4 nanoparticles) with urea containing water droplets as an aqueous phase along with cetyltrimethylammonium bromide (CTAB) and 1-butanol as a surfactant and co-surfactant. The results displayed that the prepared magnetic mesoporous silica nanocomposite has great potential for cancer drug delivery applications [58]. Fan C et al. developed oxidized mesoporous carbon nanoparticles (oMCNs) with size lesser than 200 nm and excellent water dispersibility were synthesized using a mild oxidation method. Pores of oMCNs with high drug loading efficiency (24.8% w/w) were encapsulated with resveratrol (3, 4’, 5-trihydroxy-trans-stilbene) RES. oMCNs exhibited good excellent cellular uptake and good biocompatibility. PARP and Caspase-3 protein cleavage in triple-negative breast cancer (TNBC) cell line mediated, in vitro cytotoxicity assay and apoptosis analysis showed that oMCNs-RES induced enhanced the cytotoxic effect and proapoptosis effect [59]. MTX-conjugated L-lysine coated IONPs (F-Lys-MTX NPs) were prepared by coating iron oxide magnetic nanoparticles (IONPs) with L-lysine and further conjugated with MTX through peptide linkage on IONPs surface. Significant anticancer effect for breast cancer cell lines was observed with F-Lys-MTX NPs [60]. Nosrati H et al. prepared and characterized for the potential therapeutic efficiency of curcumin (CUR)-loaded dendrimer-modified citric acid-coated Fe3O4 NPs (F@Cit@PAMAM@CUR NPs). Polyamidoamine (PAMAM, generation G5) was used to encapsulate citric acid-coated Fe3O4 nanoparticles. F@Cit@PAMAM@CUR NPs showed a suppression effect better than free curcumin on a human breast adenocarcinoma (MCF-7) cell lines [61]. Manjili HK et al. synthesized five series of mono methoxy poly U. Hani, et al. Journal of Drug Delivery Science and Technology 56 (2020) 101505 4

(ethylene glycol)-poly (e-caprolactone) (mPEG-PCL) diblock copolymers. The structure of the copolymers was characterized by H NMR, FTIR, DSC, and GPC techniques. Singel steps the nano-precipitation method was adopted to encapsulate curcumin, within micelles to get CUR-loaded mPEG-PCL(CUR/mPEG-PCL) micelles. In vivo results showed that multiple injections of CUR-loaded micelles could prolong the circulation time and increase the therapeutic efficacy of curcumin, Hence, mPEG-PCL micelles provided a convenient and appropriate system for delivery of CUR to breast cancer cells [62]. Nosrati H et al. synthesized mono methoxy poly (ethylene glycol)-poly (ecaprolactone) (mPEG-PCL) di block copolymers. In vitro anticancer activity of MTX, mPEG–PCL polymersomes and MTX-loaded polymersomes, when MTX was encapsulated with the mPEG–PCL polymersomes, the total inhibited concentration was decreased than free MTX [15]. To enhance the efficacy of BC therapy and to diminish toxicity, various novel drug delivery systems have been developed and consistently researched nowadays, a few of which are mentioned in Table 1. 3.2. Dendrimer For transportation of anticancer drugs into tumor cells and to obtain the controlled release of the delivered drug molecule the perspective nanocarriers called ‘dendrimers or dendritic polymers’ can be used [63]. These are the best-known group of NPs consisting of a core and several branches; which can be used as a carrier for diverse types of molecules. The branched structure is responsible for effective protection against the premature release of the drug into the circulatory system. Targeted therapy can be achieved by modifying the surface of dendrimers using site-specific ligands and monoclonal antibodies. It is a well recognized fact that in numerous BC cases over-expression of human EGFR-2 occurs. Hence, trastuzumab, a recombinant and humanized monoclonal antibody can be directed against this receptor for effective management of BC [64]. Humanized anti-human EGFR-2 antibody (Herceptin®) conjugated Polyamidoamine (PAMAM) dendrimers containing gold nanoparticles and gadolinium can be used for early detection and treatment of human EGFR-2-positive cancer [61,65]. Encapsulation of hydrophobic anticancer agents such as Acetyl shikonin and curcumin, in PAMAM dendrimers, results in increased solubility leading to enhanced Cytotoxicity and activity of drugs on the proliferation of cancer cells [66,67]. Cytotoxicity of trastuzumab found to be enhanced when it was conjugated to G4 PAMAM dendrimers derivatized with 30 DTPA chelators against human EGFR 2- positive BC cells [68]. Conjugate form of curcumin (obtained from the plant Curcuma longa) with dendrimer has more water solubility and capable of inducing apoptosis on BC cell effectively [69,70]. Dendrosomal curcumin can interact with NF-κB a key regulatory molecule and inhibit migration and invasion of cancer angiogenesis thereby exhibiting chemoprotective effect on BC metastasis [71]. PAMAM half-generation G4.5 dendrimer was developed containing fluorescein isothiocyanate which can enter the tumor tissue cells and can be efficiently used in diagnosis and treatment of BC [72]. G3 PAMAM-NH2 dendrimerchlorambucil conjugate have shown significantly high apoptosis in BC cells by inhibiting collagen biosynthesis than chlorambucil [73]. PAMAM dendrimers stabilized silver nanoparticles loaded with 5- fluorouracil to exhibit synergistic anticancer effect in MCF-7 human BC cell line was developed and from the results it was concluded that the Table 1 List of Nanotechnology based drug delivery systems and drugs employed for the delivery system with their application for Breast Cancer therapy. Delivery systems Drug Applications References Nanoparticles Docetaxel To carry higher levels of drug than linear polymer. [31] Simvastatin To achieve a significantly increased level of cytotoxicity. [32] Lapatinib To achieve an optimal therapeutic effect by reducing the dosage regimen. [35] Paclitaxel To deliver drug/gene/siRNA targeting. [37] Curcumin Enhanced the solubility of the drug. [33] Damnacanthal For better inhibition of cell growth. [44] Doxorubicin To get effective inhibiting proliferation and in inducing apoptosis. [46] Dendrimer Trastuzumab A recombinant and humanized monoclonal antibody as a targeted therapy. [64,65] Herceptin A humanized anti-human EGFR-2 antibody for early detection and treatment of cancer. [65] Doxorubicin To abolish drug toxicity and increases drug potency. [79,80] 5-Fluorouracil To synergistically induce apoptosis. [74] Curcumin To get enhanced solubility and cytotoxicity. [66,67] PTX To target cancer cells and get higher therapeutic efficacy. [76,77] Epirubicin To inhibit the tumor growth by targeting the tumor and providing pH-dependent drug release. [86] Docetaxel To reduce both weights and volume of the tumor cell and thereby efficiently increased killing of cancel cells. [88] Gemcitabine To get desirable tumor penetration and accumulation and greater anticancer activity. [84] Micelles PTX To get higher toxicity in comparison with taxol. [95] Curcumin To get a synergistic effect. [93] Doxorubicin To reverse multidrug resistance. [94] Liposomes PTX To improve chemotherapeutic efficiency [96] Oxaliplatin To enhance MT-3 breast cancer treatment and metastases in a mouse xenograft [97] Rapamycin To develop Human EGFR 2-positive breast cancer cells cytotoxicity [98] Gemcitabine To enhance cytotoxicity [99] Doxorubicin To build up productive delivery of the drug to EGFR2 overexpressing cells [100] PTX and Resveratrol To overcome multidrug resistance [101] PTX and Rapamycin To enhance the therapeutic effect and to limit pharmacokinetics drawbacks [102,103] Raloxifene To get effective inhibition against tumor responsible MMP-2 enzyme and angiogenesis initiation. [105] Sinitinab and Vinorelbine To get targeted delivery [106] Epirubicin and Quinacrine To exhibit high inclusive anticancer efficacy and to prevent the degeneration from VM channels after chemotherapy [107] Salinomycin & Doxorubicin To target cancer stem cells [108] Mitoxantrone To target the plasma membrane, to enhance anti-cancer activity and to decrease drug toxicity [109] Anastrozole To treat breast cancer in advanced stages in post-menopausal women [110] Exosomes Adiramycin To offer greater drug resistance via delivery of miR-222 as a transport mechanism [120] Docetaxel To controls the cell apoptosis via the transfer of specific miRNA intercellular [130] Hydrogels Cisplatin To enhance antitumor activity against MCF-7 BC cells and human colorectal cancer cells [155] Taxol To get targeted delivery and higher in vitro antitumor efficacy [156] PTX To enhance in vitro and cytotoxic effects of PTX [157] Docetaxel To enhance oral bioavailability [8] U. Hani, et al. Journal of Drug Delivery Science and Technology 56 (2020) 101505 5

prepared formulation is promising to obtain 7 simultaneous delivery 5- Flurouracil and dendrimers stabilized silver nanoparticles [74]. Advanced nanomaterials such as carbon dots can be used for bioimaging of cancer cells. Self assembled fluorescence hybrid was formed by combining carbon dots having anionic terminus and cationic acetylated G5 PAMAM dendrimer via noncovalent interaction which was found to be a potential candidate to attain drug delivery and simultaneous imaging in cancer cells [75]. A conjugate of PTX with a hydrophilic macromolecule i.e, PAMAM dendrimer through a biocleavable linker (cathepsin B-cleavable tetrapeptide Gly-Phe-Leu-Gly) to target cancer cell can effectively release the anticancer agent at the site of action and spare the normal cells thereby preventing the toxic effect on normal cells [76]. Whereas PTX loaded PEG linear dendritic copolymer-based micelles have also shown higher therapeutic efficacy on MCF-7 breast-tumor bearing mice when compared to Taxol [77]. A multifunctional PAMAM can tightly encapsulate hydrophobic drug such as 10-hydroxy camptothecin via simple complexation and can target the drug to tumor cells efficiently [78]. DOX loaded integrin mediated dendrimer gelatin nanoparticles an impressing multi-stage drug delivery system was developed for cancer treatment which can intelligently shrinks its size from large to small in the presence of matrix metalloproteinase-2 (MMP-2) and leads to higher penetration efficiency better retention in tumor cells [79]. DOX was effectively encapsulated in self assembling amphiphilic dendrimer which is having large void space for drug accommodation. The resulting nanomicelles can significantly abolish drug toxicity and increases drug potency, resulting in a promising drug carrier in cancer therapy [80]. Internalized Dendrimer pthalocyanine-encapsulated polymeric micelle resulted in a unique photochemical internalization property within the cell and thus facilitating the release of DOX to nuclei from endolysosome after photo-irradiation [81]. Kaminskas et al., have investigated the tumoricidal activity of dendrimer and liposomes in mouse and rat tumor model and concluded that DOX-conjugated PEGylated dendrimers have similar tumoricidal activity when compare to PEGylated liposome but dendrimers have shown lesser systemic toxicity [82]. For cancer therapy folate conjugated Poly-L-lysine dendrimers can be a competent carrier for DOX which have shown greater antiangiogenic and anticancer activity by providing pH-sensitive drug release and increased cellular uptake [83]. Thermoresponsive branched oligo ethylene glycol (OEG) water-soluble dendrimers can overcome the shortcomings of the currently available dendrimers. OEG dendrimer conjugated with gemcitabine due to longest peripheral of PEG segments exhibited desirable tumor penetration and accumulation and resulted in extensively greater anticancer activity compare to GEM conjugated PAMAM [84]. Thermoresponsive branched OEG water-soluble dendrimers were synthesized by Abdel et al., showed cytotoxicity against MCF-7 BC cells [85]. DNA dendrimers have excellent stability and high drug loading capacity which makes DNA nanostructure a unique candidate for biological applications. By targeting the drug to its site, side effects of the drug can be minimized and the efficacy of the drug can be enhanced. Epirubicin usage is limited due to its cardiotoxicity. Aptamer based dendrimer epirubicin complex can effectively inhibit tumor growth by targeting the tumor and providing pH-dependent drug release [86]. In aggressive BC TWIST1 a transcription factor is overexpressed which is a master regulator of cellular migration by epithelial-mesenchymal transition, siRNA based dendrimer complex to inhibit TWIST1 may provide adjunctive therapy for a patient with triple-negative BC [87]. Dendritic copolymer H40 poly (D, L-lactide) nanoparticles loaded with docetaxel when delivered along with autophagy inhibitor such as chloroquine can efficiently increase the killing of cancer cells in vitro and reduce both weight and volume of tumors in mice [88]. For targeting computed tomography imaging of BC Arg-Gly-Asp-DPhe-Lys peptide-modified PEGylated dendrimer-entrapped gold nanoparticles can be used which enables the cancer cell to get detected by CT imaging [89]. Dendrimer based gold nanoparticles developed in vivo MR-CT imaging of MCF-7 cancer cells can be effectively used as dual molecular imaging. Angio-DOX-loaded dendrigraft poly-lysine gelatin nanoparticles designed for accomplishing high accumulation and deep penetration within the tumor tissues resulted in tumor targeting and 74% tumor growth inhibition rate in a 4T1 BC mouse model [51]. Nosrati H et al. has studied the preparation and characterization of the curcumin (CUR)-loaded dendrimer-modified citric acid-coated Fe3O4 NPs which have potential therapeutic efficiency and encapsulate citric acid-coated Fe3O4 nanoparticles with polyamidoamine (PAMAM, generation G5) [61]. 3.3. Micelle Polymeric micelles are efficient to transport poorly water-soluble anticancer drugs, enhance drug stability and also site-specificity leading to enhance therapeutic efficacy when compared to all other systems [90]. Micelles are a therapeutic agent or a carrier to deliver a poorly water-soluble drug having a size of around 5–100 nm range. It consists of surfactants having a hydrophilic head and a lipophilic tail. At low concentration, it exists as a monomer and at higher concentration results in micelles formation within a narrow concentration window after aggregation and self-assembly. Hydrophobic cancer drug solubility can be enhanced using micelles. Polymer selection plays an important role in the preparation of micelle which is based on hydrophilic and hydrophobic block polymer characteristics. Steric stability and prolonged circulation time of the drug in the body are provided by hydrophilic shell. PEG the most frequently used hydrophilic polymer which is water-soluble, low toxicity and steric protector. Polyethers, polyesters, and polyamino acids are the most commonly used polymers for hydrophobic core formation [91]. Most extensively studied carriers are polymeric micelles for poorly water soluble drug delivery. The passive accumulation of anti-cancer agents in the tumor site through leaky channels through improved permeability and retention effect is enhanced by aqueous solubility and extended blood half-life of chemotherapeutic agents. Micelles size is advantageous for passive targeting to solid breast tumors [92]. Curcumin entrapped in methoxy PEG and linoleic acid conjugated polymeric micelle has been used to produce a synergetic effect as well as for routine issues against MCF- 7 breast tumor cells. MRT and T1/2 of curcumin demonstrating its increased retention time in the body by PMs are shown by pharmacokinetic studies [93]. The major obstacles in successful cancer therapy are multidrug therapy. To overcome this, amphiphilic polymeric micelles and chemosensitizers have been used. Poly (ethylene glycol)-blockpoly (lactide) micelles for co-delivery of the chemotherapeutic drug DOX with chemosensitizer curcumin were prepared to investigate dual drug-loaded micelle (DOX + Curcumin) to reverse multidrug resistance. The emulsion solvent evaporation method was used for the preparation of micelles. (DOX + Curcumin)-Micelles display prospective applications for drug-resistant breast carcinoma [94]. PTX loaded PLGA-g-dextran micelle nanoformulations showed stronger in vitro cytotoxicity against MCF-7 cells when compared to Taxol, thereby can effectively suppress the tumor growth and the toxicity against animals [95]. 3.4. Liposomes Liposomes are spherical lipid vesicles which can be formed via accumulation of lipids interacting with each other in an effectively favorable manner using either synthetic or natural polymer. Liposomes are one of the promising classes of nanomedicines with the ability to exert site-specific chemotherapy, hence convalescing the excellence of care for cancer patients [30]. pH-responsive Trojan horse liposomes loaded with PTX were developed to improve chemotherapeutic effi- ciency and stated that pH-responsive liposomes have enhanced anticancer activity in in vivo model [96]. Orthmann A et al., prepared LRP U. Hani, et al. Journal of Drug Delivery Science and Technology 56 (2020) 101505 6

targeted oxaliplatin liposome to enhance MT-3 breast cancer treatment and metastases in a mouse xenograft and found that targeted liposomes showed twelvefold greater uptake and 2.25 fold greater transcytosis when compared with non-targeted liposomes [97]. Rapamycin loaded immune liposomes employed with trastuzumab monoclonal antibody to develop Human EGFR 2-positive breast cancer cells cytotoxicity was prepared and observed that rapamycin and trastuzumab showed similar effect in vivo experiments, particularly through immunoliposomes [98]. Gemcitabine encapsulated with Hyaluronan-conjugated liposomes to treat breast cancer stem cells were developed and found that antitumor impact was 3.3 times greater for immunoliposomal gemcitabine when compare to a free drug in the xenograft mouse model due to increased cytotoxicity and CD44 receptor focusing by HA. HA-conjugated liposomes significantly enhanced cytotoxicity [99]. Zahmatkeshan M et al., developed DOX loaded anti-EGFR2 peptide liposomal formulation to build up productive delivery of drug to EGFR2 overexpressing cells and the outcomes demonstrated that optimum Anti-EGFR2/neu a peptide with PEGylated liposome Doxorubicin (DOX) can enhance the therapeutic index and selectivity of liposomal DOX in HER 2 positive breast cancer treatment [100]. Combinational delivery of Resveratrol and PTX co encapsulated liposome were developed to overcome multidrug resistance to the in vivo breast cancer cells and found that successful inhibition against drug-resistant tumor in mice without any toxicity growth and enhance the bioavailability and the in vivo drug's retention [101]. Synergistic anti-cancer effects of PTX and rapamycin co-loaded in liposomes to enhance therapeutic effect was carried out, in order to limit pharmacokinetics drawbacks. The pegylated liposomes showed tremendous colloidal stability with slow and sustained release, liposomes are more cytotoxic than the free drugs, Targeting delivery of PTX loaded liposomes by modifying A7R peptide, to regulate the tumor growth and angiogenesis at the same time and the report confirmed xenografts in vivo study from that improved vesicle uptake with greater cytotoxicity [102,103]. Magneto-liposomes loaded with Protoporphyrin IX designed for use in photodynamic therapy as potential drug delivery system to improve bioavailability and this work established the effective use of PpIX (protoporphyrin IX)-loaded MLs for in vitro photodynamic therapy, LED light source provided energy to stimulate PpIX molecules [104]. Raloxifene liposome formulated with multilayered crystalline co-chelates for effective inhibition against tumor responsible MMP-2 enzyme and angiogenesis initiation. From in vitro and in vivo reports observed that it has greater antitumor activity [105]. Targeted liposome developed with a combination of sunitinab and vinorelbine for treating invasive breast cancer by modifying a mitochondria-tropic material resulted in greater permeability retention effect and mitochondrial targeting effect liposomes (90–100 nm) resulted in prolonged blood circulation [106]. Innovative development of targeting liposomal formulation of epirubicin and quinacrine conjugated with modified PEG polymer with for eradicating invasive breast cancer cell and the study stated targeting epirubicin plus quinacrine liposomes exhibiting high inclusive anticancer efficacy and it can prevent the degeneration from VM channels after chemotherapy [107]. Cross-linked multilamellar liposomes loaded with salinomycin and doxorubicin are co-delivered to target cancer stem cells as well as breast cancer cells and this study reveals that Cross-linked multilamellar liposomes can act as a prospective platform for combination therapy [108]. Mitoxantrone encapsulated liposomes containing short-chain sphingolipid designed to target the plasma membrane, enhance anticancer activity, cancer cell membrane modulation and decrease drug toxicity and thereby it signifies a favorable approach to develop Mitoxantrone -based chemotherapy [109]. PEGylated liposomes comprising anastrozole to treat breast cancer in advanced stages in postmenopausal women and promising result showed from in vitro and in vivo evaluation compared to pure drug [110]. To attain targeted cancer therapy and ultrasensitive magnetic resonance imaging, Tumortargeting multifunctional liposomes were designed and liposomes formulated with magnetic resonance imaging contrast agent (magnetic iron oxide nanoparticles) and anticancer drugs (mitoxantrone). This research work specifies design of mitoxantrone gonadorelin-functionalized nanomedicine permits image-guided, target-specific cancer treatment [111]. Anti-laminin receptor monoclonal antibodies combine with surface of DOX loaded liposome to target human breast carcinoma cells which have great numbers of laminin receptors and it denotes an innovative approach for selective targeting carcinoma cells, antibodycoupled liposomes doxorubicin encapsulation were efficient in inhibiting colony formation [112]. Antibody-liposomal drug conjugate developed with DOX to target EGFR2-overexpressing cancer cells, along with cyclophosphamide administration can increase subsequent liposomal delivery and antitumor activity without affecting non-target tissues and this study made platform for cyclophosphamide a clinical evaluation to enhance the deposition of liposomes [113]. Ligandmodified liposomes formulated with two different anti-cancer drugs DOX and vincristine to target tumor and tumor vasculature endothelial cells for treating EGFR2-positive breast cancer and revealed the therapeutic potential of a dual-targeted drugs method against EGFR2-positive cancer cells [114]. Adriamycin liposomes conjugated with antihuman alpha-fetoprotein monoclonal antibody developed to inhibit tumor growth and enhance anti-cancer activity and better report showed by conjugated liposomes compared to unconjugated liposomes and Adriamycin distribution to heart was decreased in liposomal form without toxic side effects [115]. 3.5. Exosome An exosome is a nanosphere having a bilayered membrane consisting various types of lipids and proteins which are nonimmunogenic in nature because of similar composition as body own cells and can be used to deliver drug efficiently [116]. Multiple Micro RNAs incorporated exosomes designed to act effectively against various diseases using molecular beacons. MCF-7 was used because of the presence of higher levels of multiple micro RNAs, proved as effective Simultaneous and multiplexed detection of micro RNAs in a whole exosome for diagnosis of cancer diseases [117]. A naphthoquinone derivative of shikonin extracted from the lithospermum showed anticancer activity against tumors. Shikonin loaded exosomes developed to act against cancer cells which regulate the growth of the MCF-7 cells by reducing tumor-derived exosomal miR-128 and reduces the proliferation of tumor cells [118]. Taxane incorporated exosomes were evaluated for anticancer activity by proteomics technology as pharmacogenetic marker in chemotherapy using mass spectroscopy. The results reported that this biomarker assay showed potential activity and severity of peripheral neuropathy [119]. Adiramycin loaded exosomes developed and drug resistance studies were performed. The results stated that exosomes offered greater drug resistance via delivery of miR-222 as a transport mechanism [120]. Exosomes show multidrug resistance and act as a junction to transfer information between the cells; which cause changes in chemoresistance of tumor cells by releasing β-elemene via microRNAs [121]. Finding the microRNAs in exosomes using nanosized oligonucleotide by fluorescent technique became a modern method in the analysis of diseases by single-step process. Streptomycin improved permeability and delivery of molecular beacons enhanced target miRNA signals [122]. The communication between the microenvironment and tumor cells and the transfer of circulating endothelial miRNAs into the cancer cells attributed to the anticancer activity via exosomes by changing gene expression. It was found that enhanced Plasmatic miR-503 levels observed after chemotherapy due to enhanced miRNA levels in endothelial cells [123] that shown Fig. 4. A combination of Guggulsterone and bexarotene encourages the exosomes released by higher levels of ceramide which is a sphingolipid; it further elevates the levels of BCR protein to 20%. The results stated that this combination enhances the retention of doxorubicin and causes cell death and it was found useful in stem cell therapy [124]. The endosomal biomolecules exosomes showed a strong connection with the U. Hani, et al. Journal of Drug Delivery Science and Technology 56 (2020) 101505 7

autophagy and serve as a vesicle to communicate with the neoplastic cell and normal cell and reported the hopeful results in the treatment of cancer stem cell therapy by joining with normal cells and tumor cells. The residents of exosomes found useful as biomarkers in the treatment of cancer [125]. Phosphatidylethanolamine was recognized as an ideal lipid material of U251-MG which causes the transfer of glioblastoma exosomes into their parental cells, fibrosarcoma and breast tumors proved as effective in targeted delivery of drugs [126]. A protein miR- 10B loaded exosomes travel through different cell lines by direct uptake were found in BC cell causes exosomes secretion. The inhibitor of ceramide reduces the secretion and this route paid greater attention in the development of newer drugs [126]. Transfer of exosomes from stroma to BC cells controls the resistance to various pathways in therapy utilizing juxtacrine and paracrine signaling in chemotherapy leads to tumor growth [127]. Docetaxel loaded exosomes showed greater resistance to chemotherapy due to the delivery of biomolecules of specific miRNA [128]. Proteomic profiles of exosomes have shown more effective against tumor cells of BC via oxygenation mechanism to avoid hypoxia in the stromal environment surrounded the tumoral cells through various intercellular pathways [129]. A sensitive BC cell acquires the resistance from drug-resistance tumor cells by discharging exosomes. It was recognized in docetaxel and adiramycin exosomes control the cell apoptosis via the transfer of specific miRNA intercellular [130]. Survivin, an exosomal protein found in BC patient's serum serves as a diagnostic marker, which inhibits the cell apoptosis at various stages of cancer [131]. Epigallocatechin derived exosomes inhibits the growth of cancer cells by hindering tumor penetration with macrophages bypassing specific miRNA [132]. Stem cells derived from Human adipose tissue enhance the migration of exosomes via a novel mechanism called the Wnt signaling pathway [133]. 4. Other delivery systems 4.1. Microparticles One of the widely used technologies to target drug, to improve bioavailability and stability and to provide prolonged release of the drug is microencapsulation [134]. Use of Microparticles for breast cancer treatment is advantageous as it reduces the toxicity of the drug to non-targeted cells and tissues and provide augmented effectiveness [135]. PLGA-based microparticles loaded with bacterial-synthesized prodigiosin were synthesized by single emulsion solvent evaporation technique. Prodigiosin induces localization and apoptosis along with the reduction in viability of cells, the prepared microparticles result in the controlled delivery of drug which can be used to prevent the regrowth following surgical resection of triple-negative BC [136]. The effect of microparticle surface modification on interaction with normal and cancerous human breast epithelial cell lines were evaluated by Patino et al. Different response between two cell lines regarding cellular uptake efficiency, endocytic pathway and cytotoxicity was observed representing the significant role of surface modification of microparticles for specific cell 11 targetings [137]. DOX loaded calcium carbonate hybrid nanoporous microparticles synthesized by Bosio et al. can extensively target cancer cells and provide controlled release of loaded drugs [138]. Davoodi P et al. fabricated double-walled microparticles encapsulated with cisplatin and PTX by adopting coaxial electrohydrodynamic atomization technique and embedded into alginate-branched polyethyleneimine hydrogel injectable, in order to achieve localized dual-drug delivery and controlled release of anticancer drugs with greater anticancer response [139]. Tumor-derived tissue factor expressing microparticles represent as biomarkers which help in inhibiting and treating venous thromboembolism complications in cancer patients [140]. Hydrogel microparticle based suspension arrays for quantitative and high-throughput detection of serum levels of angiogenic markers and it has essential allegations in early detection and therapy monitoring in cancer treatment [141]. PLGA microparticles encapsulated Tumor lysates vaccination therapy developed to reduce spontaneous lung metastases in murine 4T1 adenocarcinoma breast tumors can be effective and well-tolerated in the clinic [142]. Shahanj K et al., studied injectable sustained-release curcumin microparticle as an innovative method to fight against breast cancer and found that treatment with curcumin microparticles showed a significant effect on tumor angiogenesis [143]. PLGA-based biodegradable anastrozole microparticles were formulated using emulsion/extraction method and this study revealed that microparticle drug carrier could be the better method for sustain anti-cancer treatment [144]. Magnetic iron-carbon composite microparticles formulations were also found to improve chemotherapeutic action of the drug [145]. Nonsteroidal antiestrogen tamoxifen crosslinked by alginate/chitosan microparticles was designed to target the lymphatic system for treating metastatic estrogen receptor-positive breast cancer and confirmed by in vitro characterization [146]. CD95 modified PTX-loaded PLGA microparticles enhances intercellular uptake and delivery in breast cancer cells in vitro and it has shown a greater effect than conventional PTX therapy [147]. Microparticle incorporates nucleic acids to overcome multidrug resistance and demonstrated for both hematological and nonhematological malignancies [148]. 4.2. Hydrogels Hydrogels are comprised of hydrophilic polymers dispersed into the water forming a polymeric mesh concealing drug molecules inside, and has a tendency of swelling for release of drug outside these polymeric meshes for disintegration and dissolution [30]. Hydrogels possess remarkable advantages such as higher hydrophilicity, high drug loading capacity, size flexibility, biocompatibility and ease of handling and production [149]. Poly [N-isopropylamide] incorporated hydrogels were developed using co-polymers of P(NIPA) conjugated Acrylamide and Butyl Methacrylate and homo-polymer of P(NIPA) by weight gain experiment. Swelling characteristics because of the ingestion of prodigiosin were compared with controlled environment swelling [150]. AhR ligands of 5F203 and GW 610 benzothiazole hydrogels showed antitumor activity due to the development of DNA adducts exhibited double strand in fatal DNA. In vitro studies and clinical findings suggested designing prodrugs which showed high nanomolar potency [151]. Shape coded hydrogel microparticles revealed the determination of angiogenic markers in serum to monitor various clinical disorders. Bio inert PEG hydrogels showed improvement in the sensitivity of the method by eliminating the proteins for the detection of VEGF, FGF, and PDGF in single and multiplexed assays [141]. In vitro three-dimensional culture biomimetic models (3D) of glycosaminoglycan-based hydrogel were designed which mimics in vivo microenvironment to study antitumor efficacy. The results revealed that these models showed less Fig. 4. Role of microenvironment showing the circulating endothelial miRNAs into the cancer cells. U. Hani, et al. Journal of Drug Delivery Science and Technology 56 (2020) 101505 8

sensitivity to chemotherapy than 2D cultures [152]. PEGylated fluorocarbon nanoparticles combined with PEGylated hydrocarbon nanoparticles synthesized as a thermosensitive, injectable and multicompartment hydrogel. In vitro and in vivo model suggested that peritumoral injection of drug cocktail loaded MCH formulation showed greater attention for sustained delivery of bioactive agents [153]. Three-dimensional cultures of alginate core-shell hydrogel beads developed to hinder the MCF-7 BC cell proliferation are estimated by standard proliferation assay. It has given greater attention to the drugs which have a high multicellular resistance index [154]. Thermoreversible chitosan/beta-glycerophosphate hydrogels conjugated with cisplatin showed enhanced antitumor activity against MCF-7 BC cells and human colorectal cancer cells [155]. Taxol loaded self-assembly molecular hydrogels formulated with estrone and Arg-Gly-Asp (RGD) peptide showed targeted delivery and higher in vitro antitumor efficacy [156]. PTX loaded [2,6-di-O-metyl] β cyclodextrin inclusion complex hydrogels were developed by the cold method. Assembly of mucoadhesive polyethylene oxide and thermosensitive pluronic F127 polymers showed enhanced in vitro and cytotoxic effects of PTX [157]. Nucleolin –targeting hydrogel nanoparticles developed using a copolymer of 2-carboxyethyl acrylate and acrylamide. The negatively charged modified peptide F3 combined with nucleolin using copper catalyzed – alkyne click reaction which binds to cancer cells showed higher drug loading and slow release of drugs like DOX [158]. Ionic cross-linked hydrogels developed to accelerate the release of biomolecules that provides enhanced drug release and also reversible selfhealing digitally by using ultrasound. The results suggested this technique as effective which serves as time-controlled delivery of drugs in chemotherapy [159]. Poly (methacrylic acid) incorporated pH-responsive natural hydrogels capsules can act as shape responsive carriers operated by chemical stimuli. It mimics as red blood cells by using their size, shape and provides a new approach for drug delivery [160]. Docetaxel incorporated micelles as pH-triggered hydrogels showed a greater diffusion rate in simulated gastric and intestinal fluids. The results upon pharmacokinetic data reported that docetaxel loaded micelles enhanced oral bioavailability [8]. PEG loaded chitosan thermoreversible hydrogel developed as tumor microenvironment to study in vitro cancer biology studies. The results reported the enhanced cancer cell resistance to chemotherapy, more aggregation of tumor cells and enhanced discharge of growth factors like cytokines [161]. 4.3. Microbubble Microbubbles (MBs) are increasingly being recognized as promising contrast agents for oncological molecular imaging with ultrasound. Microbubbles are bubbles smaller than 1 mm in diameter, but larger than 1 μm. Microbubbles along with low-intensity pulsed ultrasound developed to increase the curcumin cytotoxicity involving apoptosis induction and inhibition of cell motility in human BC cells and this work stated that combined therapy of microbubbles and low-intensity pulsed ultrasound has an effective approach to enhance curcumin antitumor effect [162]. Park DH et al. evaluated the adenine nucleotide translocase-2 short hairpin RNA for antitumor effects using polyethyleneimine intraperitoneal injection by adopting an ultrasound gene therapy method with microbubbles and observed greater anti tumor effects in vivo [163]. Ultrasound-mediated stimulation of microbubbles and radiation treatment investigated against breast tumor and effects on the blood vessel endothelium and the result suggested greater antitumor and antivascular effects can achieve with combination therapy of ultrasound and radiation [164]. Escoffre JM et al. evaluated doxorubicin combined with microbubble-assisted ultrasound to estimate applicability and administration effectiveness in BC cells and reported that enhanced BC cell death and glioblastoma cells with induced apoptosis can be achieved [165]. PTX loaded liposomes conjugated to the microbubble complexes were prepared for ultrasound-triggered targeted chemotherapy against BC cells and greater release succeeded with ultrasound exposure and achieved significant enhancement in PTX antitumor efficacy [166]. Ultrasound stimulated microbubbles loaded with anticancer agents combine with antiangiogenic metronomic chemotherapy to bring antivascular effects and this investigation revealed that combined Ultrasound stimulated microbubble treatments and metronomic cyclophosphamide exhibited significant tumor growth inhibition [167]. Sorace AG et al., Comparative study of Microbubbles loaded with doxorubicin and PTX for enhanced drug delivery to tumors cells when triggered with ultrasound and reported that PTX loaded ultrasound contrast agent has 20 times greater potential than doxorubicin to attain a sustain and targeted drug delivery [168]. LyP-1 peptide coated PTX loaded lipid microbubbles formulated and applied to ultrasound-assisted chemotherapy to target breast tumor cells has considerably enhanced antitumor activity [169]. Tumor angiogenesis molecular imaging using microbubbles ultrasound contrast agent was designed for sensitive recognition of intravascular targets and it was evaluated in a BC mouse model in vivo and results revealed the possibility of molecular ultrasound imaging having a low mechanical index [170]. Ultrasonic microbubbles binding LyP-1 used as bio-acoustic markers or drug carrier vehicles for targeting cancer cells within a microfluidic system can provide a high effectual in vitro approach for studying microbubble-cell interaction [171]. Biotinylated bleomycin conjugated with streptavidin-derivatized microbubbles were developed for carbohydrate dependent targeting of cancer cells and observed that bleomycin-microbubble conjugate targeted only cancer cell without affecting or binding to normal breast cell line [172]. Comparative study of sonoporation effect of two phospholipids-based vectors-liposomes and phospholipids microbubbles on the cultured cell membrane and determined that microbubbles seemed to be an innovative approach for drug and gene delivery and which could increase sonoporation effect [173]. Echogenic phospholipids-based gas-filled microbubbles by ultrasound activation were investigated for enhancing transferring antisense oligodeoxynucleotides and observed better transfection efficiency under proper ultrasound conditions [174]. Microbubbles combined low-intensity pulsed ultrasound prepared to improve curcumin cytotoxicity and drug delivery to human BC cells and found that it can markedly improve antitumor effect and this combined treatment is a promising method for antineoplastic treatment [162]. PTX-loaded nanoemulsion converted 14 microbubbles activated by ultrasound were formulated to attain controlled and targeted tumor chemotherapy and this work result indicates enhanced chemotherapeutic action with drug targeting achieved by mechanical or thermal action of ultrasound [175]. 4.4. Phytosomes A pure phospholipid complex with a pure active principle (active compounds) is called phytosome [176]. There are several applications of phytosome recently; Sabzichi M et al. applied phytosome based drug delivery of luteolin on carcinoma cells to suppressed the Nrf2 medicated signals of doxorubicin [177]. El-Far SW et al. described the phytosomal based co-delivery of drug (resveratrol) and monascus yellow pigments for the treatment of BC [13]. Hashemzehi M et al. developed a curcumin antagonistic effect to cell growth and migration that induced by thrombin through kinase inhibitor in BC [178]. 5. Therapies 5.1. Gene therapy The delivery of genes to the tumor cells in the body is known as cancer gene therapy. When genes enter the cancer cell in a patient's body they will produce an effect directly or indirectly [179]. It is a technique to correct malfunctioning genes that support disease development. In gene therapy, disease-causing genes are replaced by inserting normal genes [180]. Strategies for targeting underlying genetic U. Hani, et al. Journal of Drug Delivery Science and Technology 56 (2020) 101505 9

lesions are oncogene inactivation and replacement of tumor suppressor genes in the tumor cell [181]. Androgen receptor is activated by Lin28A due to c-myc regulation and ER-/EGFR2+ breast tumor promotion. The stimulation of the enrolment of c-Myc to the AR gene supporter was shown by ChIP analysis. Growth capacity, colonies skill, cell propagation activities, invasive capacity, and cell apoptosis inhibition was enhanced by Lin 28A in ER-/EGFR2+BC cells [182]. Stem cell gene therapy was proposed as a novel method that utilizes the pathophysiologic process of invasion of inflammatory cells into the breast tumor for controlled tumor stroma degradation. It includes modification of hematopoietic stem cells and their succeeding transplantation into tumor-bearing mice. Inducible intratumor expression of relaxin was shown after tumor cells transplant containing a relaxin gene with a vector of relaxin-showing lentivirus [183]. A protein transduction domain or cell-penetrating peptides can enable the systemic delivery of bioactive macromolecules into living cells [184]. Use of human fatty acid synthase promoter has become a new perspective as gene therapy for BC treatment. Highly expressed Fatty acid synthase is linked with malignant phenotypes and poor prognosis in invading BC. In three breast tumor cell lines, FAS expression was evaluated and analysis of cloned 680 bp-FAS promoters transcriptional activity was done in this study. Recombinant adenovirus was made and assessed its toxicity in vivo and in vitro which was driven by human FAS promoter carrying herpes simplex virus against the ganciclovir combined cell lines of human breast tumor. Varying expression of FAS was shown in BC cell lines (SK– Br3_/MCF-7_/MDA-MB-231, respectively) but relative high transcriptional activities can be initiated by FAS promoter in three kinds of cancer cell lines than normal fibroblast [185]. 21-gene RT-PCR assay effect and outcomes on adjuvant therapy in stage I BC patients was done by deriving reoccurrence score (RS) from chain reaction assay of 21 reverse transcriptase-polymerase chain. RS stratifies first stage estrogen positive, EGFR-2 normal breast tumor into three categories as low intermediate and high on the basis of 10-year distant. As a result, PT1b breast tumor patients had shown limited RS value addition [186]. Tumor vulnerability genes p53 and BRCA1 are the genes mutated or deleted in cancer gene correction strategies. Selective targeting without significant non-tumor cells toxicity is an opportunity given by this strategy. Cancer cells are inhibited by both p53 and BRCA1 that lacks mutations in these genes. Patient trials of these genes showed the potential effects of this strategy [187]. Viral etiology of human BC and human papillomavirus relationships were determined and possibilities for novel molecular targets for gene therapy and vaccine development were studied. The amplification and sequencing of HPV18 oncogenes E6 and E7 from SKBR3 cells was done. HPV18 E6 positive 6.58% and HPV18 E7 positive 23.68% were found. Propagation, metastasis, cell cycle propagation, and cell cycle-related proteins were inhibited by knockdown of HPV18 E6 and E7 in cell culture model [188]. CXCR4 bioinformatics analysis showed differential expression of 396 (chemokine receptor CXCR4) genes where 165 were up-regulated and 231 were down-regulated in the tissues having BC bone metastasis. The basis for occurrence and diagnosis of BC bone metastasis, evaluation of prognosis and target gene therapy can be provided by this analysis [189]. 5.2. Radiotherapy High energy X-rays are being used to treat cancer by radiotherapy. A linear accelerator is a machine that produces high energy rays that harm and demolish tumor cells within the area being treated. Normal cells are also affected in the area being treated, which can recover better than cancer cells. To have the maximum effect on the cancer cells, treatments are given regularly over some time. This also limits the damage to the normal cells. BC requires multi-therapy, which involves surgery and systemic therapy. At every stage of BC radiotherapy play the main role in the treatment of tumor [190]. Radiation therapy improves local control and survival, spares patient's morbidity and distress of local recurrence and also prevents seeding and reseeding of distant metastases [191]. BC women can be treated with radiotherapy in various situations such as post breast-conserving surgery, post-mastectomy or if cancer has spread to other body parts like the brain or bone [192]. Breast radiotherapy includes external beam radiotherapy [EBRT], internal radiation brachytherapy and combined EBRT-BT. Brachytherapy is widely used which comprises 82% for radiotherapy. In an external beam, radiations originate from the machine outside the body whereas in brachytherapy a radioactive source is administered inside the body for a short time period for the treatment. External beam radiation includes Hypofractionated radiation, Intraoperative radiotherapy and 3Dconformal radiotherapy [193]. After BCS, radiotherapy must be systematically performed on the entire breast for the prevention of local recurrence. Radiation is given after surgery once the tissue has been healed or if the dose of chemotherapy is completed. Once ductal carcinoma in situ is conformed, adjuvant radiotherapy must be performed after lumpectomy. After a mastectomy, and if there is an axillary nodal involvement, whatever the number of involved lymph nodes chest wall irradiation is required for pT3-T4 tumors. Dose to the chest wall or the breast must be between 45 and 50Gy with conventional fractionation. A higher dose is required if the patient is younger than 60 years old [194]. Intraoperative radiation therapy allows the delivery of radiation to the tumor bed directly during surgery [195]. Tiffany et al. performed a retrospective review on postoperative outcomes of patients with external beam radiation therapy exposure and observed a significantly higher risk of complication in patients [196]. Rocco et al. conduct a study on intraoperative radiation therapy with electrons in conservative treatment of BC and suggested that this can be a suitable therapy for low-risk patients [197]. Radiation therapy generally causes teratogenicity, induction of childhood malignancies and hematological disorders in pregnant women occur between 15 and 50 days during organogenesis [198]. Pregnant patient with BC was investigated for fatal doses. An increase in fetal dose by 70% and 40% was confirmed respectively for 3-D conformal radiotherapy and intensity-modulated radiation therapy when it was closed by 5 cm to the irradiated field [199]. Roghayeh et al. Conducted a study on calculating the absorbed dose by the thyroid in BC radiotherapy using Monte Carlo Nuclear Particles Code (MCNP-4C) and determining the scattered radiation dose absorbed by thyroid as one of the radiosensitive tissues in the human body to determine absorbed dose in BC patients undergoing radiotherapy. The measurement showed that 10% of all received dose was absorbed by the thyroid, while the result of calculation showed that only 9% of that was absorbed by thyroid which is 1% less with respect to measured values [200]. Isabel J et al. determined the median cost of radiotherapy using a multivariable Fine-Gray model as $8100 [201]. Ubukata et al. have described that recall dermatitis due to radiation occur after 6.4 years of breast-conserving surgery in an old women of 37 years ago. Eczema and erythema were found at the irradiated site [202]. Kennedy et al. studies lead to the novel hypothesis that radiation exposure in humans' leads to acute radiation syndrome-like Coagulopathy induced by radiation and associated hemorrhage which is the major cause of death. To prevent adverse biological radiation-induced effects anti-histone antibodies might be helpful as therapeutic agents that can block the lethal effects of elevated circulating nucleosome/ histone levels in blood [203]. The study related to QOL reveals that radiotherapy did not worsen QOL in BC patients during and until oneyear post-radiotherapy treatment. However, the pre-radiotherapy patients may experience decreased physical and mental function during and up to one-year post-radiotherapy [204]. 6. Oral delivery Oral delivery in cancer therapy has been principally influenced patients with the quality of life and reduced health care costs. Simultaneously, challenges do prevail in the oral therapy of drug-like agents such as oral delivery of macromolecules includes insulin-like U. Hani, et al. Journal of Drug Delivery Science and Technology 56 (2020) 101505 10