Recent advances in folic acid engineered nanocarriers for treatment of breast cancer

Journal Pre-proof Recent advances in folic acid engineered nanocarriers for treatment of breast cancer Priti Tagde, Giriraj Kulkarni, Dinesh Kumar Mishra, Prashant Kesharwani

PII:

S1773-2247(19)31989-6

DOI:

https://doi.org/10.1016/j.jddst.2020.101613

Reference:

JDDST 101613

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 16 December 2019 Revised Date:

19 February 2020

Accepted Date: 20 February 2020

Please cite this article as: P. Tagde, G. Kulkarni, D.K. Mishra, P. Kesharwani, Recent advances in folic acid engineered nanocarriers for treatment of breast cancer, Journal of Drug Delivery Science and Technology (2020), doi: https://doi.org/10.1016/j.jddst.2020.101613. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

Graphical abstract:

Recent advances in folic acid engineered nanocarriers for treatment of breast cancer

Priti Tagde1*, Giriraj Kulkarni1, Dinesh Kumar Mishra2#, Prashant Kesharwani3*

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Amity Institute of Pharmacy, Amity University Noida, U.P., India 2 Indore Institute of Pharmacy, Indore (M.P.) 3 Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, 110062, India

*Author for correspondence: *Dr. Prashant Kesharwani Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow, UP, 226031 India Email: [email protected] [email protected] Tel./Fax: +91-7582-244432

#Dr. Dinesh Kumar Mishra Associate Professor Indore Institute of Pharmacy, Indore (M.P.) India Email: [email protected] Tel./Fax: +91-9826345725

Disclosures: There is no conflict of interest and disclosures associated with the manuscript.

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Abstract Breast cancer is one of the most prevalent types of cancer among female patients, which is caused by mutations in the oestrogen/progesterone receptors. Conventional treatment methods are generally used for eradication of cancer; however, it has been associated with numerous adverse effects which emerge the need of nanotechnology in cancer therapy. This review is focused on nanotechnology based-therapeutic approaches increasingly acceptable to improve therapeutic efficacy and reduce unwanted effects associated with anticancer payload in breast cancer. Among several nanocarriers, folic acid conjugated nanocarriers have shown remarkable targeting efficiency due to their promising specificity for folate receptors overexpressed on the surface of breast cancer cells. This target-specificity results in significant enhancement of drug internalization into the breast cancer cells and reduces accumulation into the off-target organs. Despite exhibiting excellent anticancer efficacy, folate-conjugated nanocarriers have also evidenced as an effective theranostic agent. This review has highlighted the potential of folate-conjugated nanotherapeutics for treatment of breast cancer, challenges pertaining to anticancer therapy, and future perspectives on the pharmaceutical significance and anticancer efficacy of folate conjugated nanocarriers. Keywords: Breast cancer; folic acid; nanocarriers; theranostic; targeted therapy; chemotherapy.

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1. Introduction Cancer is a group of diseases, in which the mutation of the body cells causes uncontrolled growth. In the initial stage of this disease is designated as a small mass or lump, and called by the name of the originating organ [1,2]. In 2018, International Agency for Research on Cancer (IARC) estimates that there were 17 million new cancer cases worldwide. We can observe that the common cancer types vary by geographic area. The eye-catching part is that in about 19 out of 20 parts of the world area, breast cancer is the most common type of cancer in female, where the women among the age of 50 years or over mostly encounter this disease [1,3,4] Based on the statistics of GLOBOCAN, about 18.1 million new cases in worldwide were diagnosed with breast cancer in 2018.This represents 12% of all new cancer cases and 25% of all cancers in women with breast cancer becoming the second leading cause of death due to cancer [5]. The statistics show that the relatively higher incidence of breast cancer was in Northern America and Oceania and the lowest incidence in Asia and Africa [6]. Majority of the breast cancer starts at the lobules (milk producing glands of the breast) and to the ducts which connect those lobules to the nipple. Uncontrolled growth of these cells results in generation of the tumor which usually can be felt as a lump or can be observed on an Xray. Upon progress, tumor mass becomes malignant when the cells invade into surrounding normal cells or metastasize (spread) to other parts of the body. [1,7] Commonly, breast cancer happens in women, yet men have the rare chances of getting breast cancer, too [8]. Treatment of breast cancer involves complete or partial mastectomy, where radiotherapy is continued following partial mastectomy or breast-conserving surgery. However, long term survival is reportedly associated with complete masectomy [9]. Chemotherapy is sometimes initiated following mastectomy to completely remove the metastatic cancer cells from the body of the patients [10]. Anticancer drugs, such as doxorubicin (DOX), docetaxel (DTX), paclitaxel (PTX), 5fluorouracil (5-FU), idarubicin and vincristine are available in the market for chemotherapy[11–13]. It has been reported that the patients who undergo chemotherapy have increased survival rate (3-9%) than the patients undergoing no chemotherapy [14]. Based on the statistics of Komen, women younger than age 50 with treatment have 55-84% in 15-year overall survival rate and women younger than age 69 but older than 50 with treatment have 62%-86% in 15-year overall survival rate [15]. Although, chemotherapy successfully

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prolongs the life-time of patients, anticancer drugs have several limitations to deliver to the patients. Due to lipophilicity of the components, oral delivery is highly restricted, thus high concentration of parenteral administration is necessary to maintain the effective concentration for longer period. This higher concentration of the drug in systemic circulation leads to serious cytotoxic effects including non-specific pharmacokinetics [16]. Due to a range of limitations brought by conventional cancer chemotherapies, such as drug resistance, nondifferentiation between healthy and cancer cells leading to severe side effects and systemic toxicity, scientists are in quest to find solutions in chemotherapy. innovative drug delivery technology has become important to obtain enhanced solubility, improved efficacy with decreased side effects by reducing the quantity of the required chemotherapeutic dosage and to make the onset of therapeutic action more rapid and prolonged [17–19]. 2. The need for nanotechnology and targeting ligand Nanotechnology is widely suggested to reduce the dosage quantity as well as frequency significantly with similar pharmacological profile and reduced adverse effects. This is due to the ability of nanocarriers to deliver within confined tissues [13,16,20–28]. Nanosystems range from carbon nanotubes, dendrimer, liposome, metallic nanoparticles (NPs), nanocrystals, quantum dots to polymeric micelles and polymeric NPs. Biodegradability, biocompatibility and drug protection are characteristics of polymeric NPs which make them useful in controlled and sustained drug delivery systems [17,29–32]. Easy synthesis, purification, functionalization, scalability and targeting ability add to preferential use of polymeric NPs [33–35]. Ligands expressed on cells play a definitive role in targeted delivery to diseased cells. Thus, determination of right targeting ligand is key for improved targeted delivery [19,36]. Ligandconjugated polymeric NPs have been used for a while and they demonstrate potential in reducing the dose-limiting toxicity. Compared to the normal breast cells, up-regulated folate receptors are found in cancer cells especially folate receptor alpha (FR-α). FR-α plays role in folate uptake, folate being a basic component of cell metabolism, DNA synthesis and repair, [34,37,38]. High binding affinity (kd = 10−10M), high susceptibility with organic and aqueous solvents, low immunogenicity, low molecular weight (MW 441.4) and low cost make of folic acid (FA) conjugated NPs make it a reasonable and feasible way to counter the tumors [39]. This review summaries folate as targeting ligand, folate targeted NP in breast cancer treatment and theranostic application of folate targeted NP in breast cancer. The specific aim

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is to summary and analysis FA conjugated NPs from several studies and clinical trials to give clinicians an alternative delivery drug system in breast cancer chemotherapy. 3. Folate as Targeting Ligand Recently folate has been studied by the researchers in chemotherapy because of its abundance in the cancer cells as it contributes to cell division [40]. Folate receptor (FR) is overly expressed in some cancer cells because of high demand of folate for cellular DNA repair during the early stage of carcinogenesis by the tumor cells for rapid duplication [40–44]. By using folate as targeting ligand for chemotherapy, the adverse effect of chemotherapeutic agents can be reduced as it will specifically bind to cancer cell overexpressed with FR and to a lesser extent to the normal human cell [43,45,46]. Hence, FR targeting medicine had been investigated as the treatment of various cancer chemotherapy included breast cancer. 3.1 Distribution of folate receptors in normal and cancer cells Basically, there are 3 sub-types of FRs available, namely FRα, FRβ and FRγ. FRα is widely expressed in various types of carcinoma cells including breast cancer whereas distributed less in normal human cells [40,41,47]. The highly expressed FRα help in the uncontrolled and rapid duplication of tumor cells [41]. Because of the high expression in a cancer cell, FRα is considered as the main targeting receptor for folate-conjugated drugs in chemotherapy [41,44]. 3.2 Structural basis for binding of folic acid to folate receptors FR interacts with folate specifically and has high affinity to mediate folate uptake into the cell form circulatory system [40,41,43,44]. FR has globular structure consisting of 4 long αhelices, 2 short α-helices and 4 β-strands as well as a loop region. It has been demonstrated that folate usually forms a strong hydrogen bond and hydrophilic interaction with FRα in the loop region [41]. The strong interactions and bonds are the key factor to indicate the high affinity of folate to FRα [40,41]. [40,44,48]. 3.3 Mechanism of folate conjugates uptake by folate receptors it is hypothesized that the folate conjugated delivery systems may specifically bind to the FR, expressed on the surface of cancer cells, and transported intracellularly via clathrin mediated endocytosis [11,42,43]. The folate conjugated carriers of chemotherapeutics in circulation get bind to FRs on the carcinoma cell surface and invagination, internalization and vesicle

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formation follow binding of the folate conjugates. When the pH inside the vesicle decreases, folate conjugated chemotherapeutic agent releases into the cytosol to produce the desired pharmacological action within the cancerous cells. This action is to stop the cell division or initiate apoptosis. folate ligand conjugates behave differently upon binding indifferent cancer cells [43,49]. After the folate conjugated chemotherapeutic agent is released into the cell, the internalized FRs re-combine to the cell surface for further endocytosis of folate-targeted drugs or free folates. The recycling of FR back to the tumor cell surface makes the folate conjugated carriers continuous internalization by the cancer cells [41,43,50] (Figure1). Therefore, advanced research is needed to develop folate as the targeting ligand for chemotherapy drug delivery system to optimize the effectiveness of anticancer drugs to tumor cells and reduce the side effect of drugs to the healthy human cells. The subsequent section of this review summarises current information on approaches towards folate conjugated NPs used in the experimental breast cancer models. 4. Folate targeted nanoparticles in breast cancer treatment Potent chemotherapeutic agents are associated with serious side effects, hence recent researches have been focused on targeted delivery of drugs via nanocarriers. 4.1 Folate conjugated nanoparticles loaded with doxorubicin Various stimuli responsive nanocarriers, such as temperature-, redox and sensitive nanoparticles are studied for targeted delivery along with targeting ligand [51]. Following this concept, multiblock copolymer nanocarrier was developed for treating breast cancer [33,52], where the developed NPs were conjugated with folate and trastuzumab through the hydroxyl group and loaded with DOX. Redox responsive behaviour of polymer NPs has been confirmed by drug release profile, where at pH 5.5, ~72% of the drug was released as compared to the pH 7.4, where the release is only ~18%. Studies about cellular uptake revealed that FA and trastuzumab conjugated NPs have higher cellular uptake along with higher apoptosis in MCF-7 cell line compared to the control group. Likewise, in vivo study indicated that ligand conjugated NPs have no significant toxicity and high tumor regression (91%) in Ehrlich ascites tumor as compared to free DOX[33] (Figure 2). Similarly, Late et al. developed ATRP-based biodegradable triblock copolymer for breast cancer treatment. To achieve dual targeting, the researchers attached AS1411 aptamer along with FA via EDCNHS coupling. Again, drug release at pH 5.0 was ~70% as compared to ~25% of drug release at pH 7.4 due to the presence of the acid-sensitive hydrazone linkage. Study about cellular

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uptake indicated that higher cellular uptake had shown by dual targeting with FA and AS1411 aptamer as compared to non-targeted or single NPs together with higher apoptosis in PANC-1 and MCF-7, whereas normal body cells are free from the adverse effect of DOX[53,54]. In another study, Hussaina et al. developed folate conjugated polymeric-gold composite NPs (GNPs) combining DOX and laser photothermal therapy. To deliver the DOX to the targeted breast cancer cells, polyethylene glycol (PEG) was engineered with the drug and modified GNPs surface with folate. By using laser light, GNPs could be photo-excited to mediate the cancer cell by hyperthermia. In vitro studies, folate conjugated DOX loaded polymeric GNPs together with laser photothermal therapy had shown superior efficacy compared to DOX alone especially on highly FA expressed breast cancer cell (MDA-MB-231) [55,56].Pramod et al. synthesized DOX conjugated with n-acetyl glucosamine or FA- functionalized mesoporous silica NPs (DOX-NAG-MSNPs or DOX-FA-MSNPs). It was observed that DOX-NAG-MSNP possess higher cellular uptake and cytotoxicity effect towards the breast cancer cell, namely MCF-7 and MDA-MB-231 cells as compared to DOX-FA-MSNP. Yet, either DOX-FA-MSNPs or DOX-NAG-MSNPs can deliver DOX drug to the targeted breast cell in a better way compared to free DOX alone [29,57]. 4.2 Folate conjugated nanoparticles loaded with docetaxel DTX is second generation, semisynthetic taxane drug, used to treat breast cancer by cellcycle arrest at the G2/M phase and cell death. Faranak et al. prepared DTX attached folate conjugated poly (L-α-glutamyl glutamine) (PGG) NP which had shown lower toxicity to normal cell due to specificity and highly targeted drug delivery toward breast cancer compared to free DTX alone. In vitro study indicates that the NPs have higher cytotoxicity owing its action to overexpressed folate receptors in cell line used (MCF 7). Higher anticancer therapeutic efficacy is evidenced by significant inhibition of tumour growth by the targeted NPs compared to DTX treatment alone[58,59]. Furthermore, Nateghian et al. developed biotin or folate-decorated human serum albumin NPs to increase the aqueous solubility of DTX. NPs have specific cellular uptake by cancer cells that overexpressed FA or biotin receptor as well as it can deliver the DTX to the targeted site. In vivo studies on tumorized BULB/c mice by 4T1 cell line, NPs showed a higher survival rate and higher reduction in tumor size compared to DTX alone [60]. 4.3 Folate conjugated nanoparticles loaded with Paclitaxel

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PTX emerged as first-line drug for breast cancer because of its high efficacy and tolerability. Nazli et al developed folate conjugated amphiphilic β-cyclodextrins loaded with PTX for breast cancer treatment. PTX being loaded within hydrophobic chains for delivery to targeted site. Due to high encapsulation within the 100 nm particles, these NPs have higher stability than the other nanoparticulate system thus it can be inferred that this formulation could be formulated to obtain sustained release of the incorporated drug and to have superior characteristics than the conventional dosage form. During the in vitro assessment, T-47D and ZR-75-1 human breast cancer cells were being used in the assessment of anti-cancer efficacy since they possess different amount of folate receptor on their surface. Higher anticancer efficacy had been reported by PTX-loaded NPs because of better interaction between folate receptor positive cancer cells. PTX conjugation with folate-conjugated cyclodextrin (FCD)-1 and FCD-2 have showed an increased sensitivity of the breast cancer cell toward the developed NPs, which increase its efficacy of cellular uptake and reduce the side effect of PTX thus resulted in increased safety profile [61,62]. Another study investigated lipid-polymer hybrid PTX carrier where the lipid shell was modified by folate (FA) and polymer-core NPs (FLPNPs) in order obtain site directed delivery, sustained and controlled release of PTX. Through hydrophobic interaction, hydrophobic PTX can be incorporated into hydrophobic core of the NPs. In-vitro studies demonstrated higher cytotoxicity at a high concentration of the drug and long exposure time of PTX-loaded FLPNPs as compared to PTX-loaded LPNPs which is not conjugate with folate. Low toxicity yet similar antitumor efficacy had showed by NPs (PTX-loaded FLPNPs) in in-vivo studies in BALB/c mice bearing EMT6 tumor via intratumoral chemotherapy compared to PTX alone. Higher cellular accumulation of PTX-loaded FLPNPs in cancerous cell indicated the importance of folate targeted NPs [63]. Thus, PTX-loaded FLPNPs provided greater inhibition of tumor growth than the non-targeted PTX-loaded LPNPs. 4.4 Folate conjugated nanoparticles loaded with other drugs The use of 5-FU, a broad-spectrum cytotoxic agent, is limited due to its non-selective distribution, short half-life and possible risk of tumor cells developing drug resistance. Considering that, Mazen et al had developed poly (D, L-lactide-co-glycolide) NPs that were PEGylated besides being decorated with FA (FOL-PEG-PLGA NPs), for the treatment of breast and colon cancer. Due to NPs characteristic, drug has shown to release in sustained (biphasic) drug release manner. FOL-PEG-PLGA NPs loaded with 5-FU had shown a 8

superior cytotoxicity effect over the non-targeted PLGA-based NPs in HT-29 colon and MCF-7 breast cancer cell lines, where both were overexpressed with folate receptors. NPs have also shown highly hemo-compatible with negligible cytotoxicity which makes them a good tool for drug carriers. In vitro cytotoxicity studies demonstrated a 4-fold less of half maximal inhibitory concentration of 5-FU-loaded FOL-PEG-PLGA NPs compared to5-FUloaded PLGA NPs [64,65].Another potential approach of magnetic NPs (MNPs) in cancer treatment by targeting the tumor site using an externally applied magnetic field was being developed and studied by Gunduz et al. for treating breast cancer. These NPs were further conjugated with the targeting ligand, namely FA and subsequently loaded with the anticancer drug, idarubicin. Successful internalization and accumulation of MNPs in the MCF-7 cells were established using light and confocal microscopy. No toxicity has been shown by empty MNPs on MCF-7 cells in the concentration range of 0–500 mg/mL. Nevertheless, a significant toxicity that is concentration-dependent has been demonstrated by idarubicinloaded PEG coated NPs. Moreover, idarubicin-loaded MNPs have shown to cause more toxicity in the comparison with free idarubicin. XTT cell proliferation assay results showed that when idarubicin is given as FA conjugated PEG coated MNPs, the IC50 value of free idarubicin decreases from 2.48 µM to 1.25 µM. In vitro study also showed that idarubicinloaded MNPs had a 2-fold higher cytotoxicity as compared to free idarubicin on MCF-7 cell line [66,67].This study was continued by Jianian et al. who developed bifunctional NPs (BFNPs) where the NPs were modified with 2 ligands simultaneously, namely the cell penetrating peptide R7 and FA, which serve to deliver chemotherapeutic agents and for breast cancer therapy. Analysis of vincristine entrapped NPs showed biphasic releasing manner of the drug, as discussed earlier. By using dynamic laser scattering method, the particle size of the conjugated NPs in BF-NPs was found to be greater than that in PLGA–PEG–folate NPs or PLGA–PEG– R7. Additionally, the cellular uptakes of targeted NPs were merely modified by FA or R7. Simultaneously, in vitro cytotoxicity studies for cell apoptosis and cell cycle arrest studies revealed a higher potency of BF-NPs towards MCF-T cells when compared to the NPs modified by FA or R7. Other than that, BF-NPs also confers another advantage that is a higher ability to induce apoptosis in tumor cells while having a very limited effect on the normal cells[68,69]. In contrast, Liwei et al. made an approach to overcome multidrug resistance, they had developed PLGA-PEG-folate and cell penetrating peptide R7-conjugated PLGA-PEG to deliver vincristine (VCR) (VCR-Fol/R7 NPs). It was demonstrated that the release of VCR is pH-dependent where the weak acidic environment favours the rapid release of a larger 9

amount of VCR. In comparison with the NPs merely modified by folate or R7, the conjugated VCR-Fol/R7 NPs were shown to enhance both cellular uptake and cytotoxicity in MCF-7 and MCF-7/Adr cells. It also can escape drug efflux mediated by P-glycoprotein due to folatemediated endocytosis and stronger intracellular penetration leading to drug accumulation in resistant tumor cells. In vivo, pH sensitivity and folate receptor mediated effect was suggested to be the dual reason for the effective nanocarrier, Fol/R7 NPs to enhance tumor targeting efficacy which eventually leads to active targeting on tumor site [70,71]. Mollarazi et al developed another targeted tool, Sm-folate-polyethyleneimine-conjugated chitosan NPs which were water soluble and biocompatible. Interestingly, binding studies revealed greater internalisation for MCF-7 cells compared to 4T1. Besides, in vivo biodistribution studies support kidney as the main route for the excretion of the NPs [72]. Similarly, curcumin loaded folate-modified-chitosan-NPs were developed and fabricated via self-assembling process, by Boroujeni et al., with targeting ability. Eventually, the releasing rate of curcumin from folate-modified chitosan NPs was dependent on the pH of the release medium, where a change of pH from 7.4 to 5.0 hastened the release of curcumin. Curcumin loaded NPs have been proven of having good potential to be a drug delivery system for breast cancer therapy [73]. Quercetin was also considered with targeting approach to overcome its limitations of low bioavailability and poor water solubility, which restricted the use of this drug in cancer therapy even though it has many useful effects. Hence, Sarkar et al. synthesized FA conjugated mesoporous silica NPs (MSN-FA-Q) as vehicles to deliver quercetin that aimed to target against breast cancer cells. The NP distribution and assess tumor regression were investigated using ex vivo optical imaging and CAM assay respectively. Results showed that MSN-FA-Q NPs have led to higher cellular intake as well as drug bioavailability to the breast cancer cells. By regulating Akt and Bax signalling pathways, MSNs not only promoted cell cycle arrest to the breast cancer cells, but also cell apoptosis besides having the antimigratory role [74]. On the other hand, Singh et al. developed non-PEGylated poly (d,llactide-co-glycolide) (PLGA) NPs and FA conjugated PEGylated and loaded them with saquinavir (SQV) to investigate anticancer potential. By comparing SQV-loaded-FAconjugated-PEGylated-poly(d,l-lactide-co-glycolide) NPs with the non-targeted SQV-loadedpoly(d,l-lactide-co-glycolide) NPs, SQV-loaded-FA-conjugated-PEGylated-poly(d,l-lactideco-glycolide) NPs were concluded to exhibit superior anticancer potential through the enhancement of cytotoxicity, cellular uptake and preferable uptake by cancerous cells [75]. To summarize, from the above reports on FA conjugated nanocarriers from several 10

researchers are evident for improved anticancer therapy with decreased toxicities of the incorporated chemotherapeutic agent. 4.5 Theranostic applications of folate targeted nanoparticles in breast cancer Breast cancer can be effectively treated/diagnosed with the emerging of nanotechnology which could be exhibiting both targeted drug delivery and theranostics properties [76]. For theranostic application purposes, various stimuli responsive nanocarriers have been studied for precise targeting, cell imaging and drug transporting functionalities (Figure 3). Multifunctional NP encapsulation of luminescent Mn:ZnS quantum dots (QDs) with chitosan and FA was developed to allow an instantaneous targeted drug transport and cell imaging. As the QDs are conjugated with chitosan, it acts as a stabilizer and provides an active binding site toward cancerous cells. Besides, incorporation of FA to the nanocarrier as targeting agent brings the emission of orange–red fluorescence at around 600nm with 15% of Mn2+ doping concentration and more stable at decreased pH as well as alkaline pH. It is proven that this assynthesized composites exhibit non-toxic properties toward human breast cell line MDAMB-231, MCF-10 and MCF-7 at concentration of 500 µg/mL. Hence, conjugated Mn:ZnS QDs are revealed to be fluorescence markers in cellular uptake studies as it improves the internalization and binding affinity of the nanocarrier toward folate receptor-overexpressed cells [77]. On similar lines, Zhou et al. reported bovine serum albumin (BSA) protected gold fluorescence nanoclusters (GNC) combined with cisplatin prodrug and FA for cell imaging and targeted chemotherapy of breast cancer. As first line anti-cancer drug, hydrophilic cisplatin MDDP is conjugated to achieve cisplatin prodrug, i.e. conjugated GNC NPs. A BSA protected GNC is more stable than QDs and provides emission of high fluorescence in a wavelength of 650 – 750nm. The particle size of less then ten nanometers allows it to have quick kidney clearance and reduced side effects in biomedical application. Not to mention FA modified GNC also quickens cellular uptake and increases cytotoxicity of GNC-Pt, which allows it to have a dual-functional nanoplatform as it plays the roles of targeted drug delivery and fluorescence imaging on cancer cells. [78] Likewise, Heidari Majd et al. considered MNPs as one of the multifunctional theranostic application. By using the method of thermal decomposition of Fe (acac)3, Tamoxifen (TMX) loaded FA-armed PEGylated MNPs are synthesized. It gives an average particle size of 40nm

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which allows a loading efficiency of 49.1%. Hence, this TMX MNPs presented a controlled deliverance of TMX in 72h with 90% release. Cytotoxicity analysis also shows a significant high binding profile which leads to inhibition of growth in MCF-7 cells however not in A549 cell line [79]. Moreover, Alibolandi et al. described QD and DOX-encapsulated PEG-PLGA NPs conjugated with folate for active cancer targeting as well as cell imaging. It exhibits amphiphilic characteristics as FA-QD-DOX-encapsulated PEG-PLGA NPs allows the delivering of both hydrophobic and hydrophilic drugs. This allows it to have a sustained release of DOX approximately for 12 days as evidenced from thein vitro experiments. Due to the folate receptor-overexpressed cells, it specifically accumulate in MCF-7 and 4T1 cells and also exhibit higher cytotoxicity in vitro. However in vivo experiments showed that the FA-conjugated-QD encapsulated NPs accumulate for 6h at tumor sites following IV injection [80]. Similarly, Majd et al. proposed the use of FA/ fluorescein isothiocyanate-PEG fluoromagnetic NPs conjugated with mitoxantrone (MTX)(Fe3O4-DPA-PEG-FA/FITC NPs) for the advance of instantaneous therapy and imaging of breast cancer.Targeted fluoromagnetic NPs are synthesized in the reaction known as thermal decomposition reaction of Fe(acac)3 at 270°C. The size of prepared NPs has ranged about 30-35 nm with properties of FR-positive recognition in MCF-7 cells. By conjugating moieties on NPs, disease specific markers are allowed to be detected, hence,resulting in agreater permeation and retention (EPR) effects as well as improved specific targeting of the tumor cells [81]. Barar et al. in the year 2015 developed FA-armed-MTX-combined MNPs. This MNPs exhibit an external magnetic field which allows it not only to accumulate but also intensifies the accumulation at the site of targeted tumor cells through active and passive targeting. It penetrates both A549 and MCF-7 cell lines with a hydrodynamic radius range of 0.8 to 6500 nm. MCF-7 cells showed IC50 values of 1.7 µg/mL and 3 µg/mL, 48 h and 24 h posttreatment, respectively. 4′,6-diamidino-2-phenylindole staining and DNA ladder assays showed a result of DNA fragmentation as well as significant condensation of the nucleus in the FR-positive MCF-7 cells. Moreover, by examining through FITC-labeled annexin V and quantitative polymerase chain reaction assays, late apoptosis (>80%) can be confirmed, in which it also shows a cytotoxic effect via modifications of apoptosis-related genes in MCF-7 cells [82].

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Super paramagnetic iron oxide NPs (SPIONs) combined with FA were projected by Huang et al. DOX loaded SPIONs allows a higher release rate in low pH PBS as it consists of a mean hydrodynamic diameter of 23, 40 and 67nm, which performs outstanding colloidal stability. Besides, by testing with nude mice, results show enhanced growth inhibition on xenograft MCF-7 breast tumor in vivo and on MCF-7 cells in vitro, whereas the vital organs of the mice do not show toxicity of DOX loaded super paramagnetic iron oxide NPs after 35-days of treatment [83]. Simultaneously, FA conjugation to the nanocarriers can also be a novel nanotheranostic approach to obtain personalized therapy with better prognosis, which will step towards the cure against this deadliest disease. 5. Conclusion Numbers of studies have been carried out to study the effect of anticancer nanomedicines which could lower the risk of side effects of chemotherapeutic drugs. Due to specific site binding affinity, apoptosis is achieved by dual targeting ability of nanocarriers and significantly inhibit tumour growth as well as reduce the volume of tumour. Moreover, surface modifying of nanocarriers allow a sustained and continuous drug release profile and higher cytotoxicity effects in folate over-expressed cell line. It also enables better internalization of therapeutic agents within the cancer cells as it avoids early inactivation of the drug during its transport. Amphiphilic properties of nanocarriers enable a sustained release of delivering of both hydrophobic and hydrophilic drugs. Desired size and shapes, magnetic and chemical properties synthesized NPs possess a promising drug loading and release efficiencies. Besides, in acidic pH condition, certain drugs are able to release faster from the nanocarriers and according to their metabolic route, accumulation occurs at different tumour sites. Conjugated with therapeutic agents, these nanosystems able to be equipped with imaging and homing techniques that allow theranostics effects. As for theranostic application, fluoromagnetic NPs provide a significant improvement on fluorescence imaging and therapy of breast cancer for specific instantaneous targeting and imaging. Particle size plays a vital role as in several to ten nanometers, it allows quick kidney clearance and also reduces side effects in biomedical application. By targeting FA conjugated nanosystems to cancer cells, it increases their tumour accumulation as co-delivery vehicles for imaging agents conjugated with anticancer drugs. However, the challenges are encountered during researches. Greatest challenge is retaining 13

the biochemical activity of NPs as some exhibit short half-life in water, hence decreases coupling efficiency on cancerous cell lines. Additionally, manufacturing associated problem such as low loading, batch to batch variation and high production cost is the bottleneck for commercialization of nanoformulation for cancer therapy. Therefore, challenges should be overcome through numerous researches and ongoing rational therapeutic goals for cancer treatment. FR-targeted drug delivery is one of the most frequently used approach as anticancer therapeutics which can be proved by number of publications. However, only few has been translated into clinic. This is because preclinical studies usually comprise use of cell lines and animal tumor models having high folate receptor expressions (KB cells). The prerequisite for clinical translation must take into account the more moderate level of FR expression found in clinical situations [84]. Very recently movement of vitafolitide and fartetuzumab into Phase III trial proposes that FR targeting is finally reaching a critical point, however, continued research is warranted.

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Figure captions: Figure 1: The diagram shows the specificity of folate as the targeting ligand binding with FR on the cancel tumor membrane. Figure 2: The diagram illustrates drug delivery by the dual targeted (folic acid and trastuzumab) redox sensitive polymeric nanocarriers composed of multiblock copolymers, in breast cancers. Release of drug (doxorubicin) is favoured in acidic environment and the presence of glutathione reduced (GSH). GSH that present in the cytoplasm accelerates drug release due to the cleavage of disulfide linkages in the polymeric nanoparticles via reversible disulfide-thiol exchange reaction. Figure 3: Folic acid conjugated theranostic nanoparticles for targeted fluorescence imaging and chemotherapy of the metastatic breast cancer. Targeted drug delivery and fluorescence images clearly demonstrated above at cancer cell site by single tail vein injection given in vivo.

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Declaration of Interest Statement: There is no conflict of interest and disclosures associated with the manuscript.