Folic acid functionalized nanoparticles as pharmaceutical carriers in drug delivery systems

2.1.1 Chitosan NPs

Targeted nanoscaled chitosan (CS)-based DDSs are the subject of interest as targeted carriers of therapeutics and diagnostics agents to cancer cells, which have improved systemic performance in cancer detection and therapy (Figure 4; Chen et al., 2008; Park et al., 2004). The CS, as natural biopolymer, has excellent biocompatibility, biodegradability, great bioavailability, hydrophilicity, and low toxicity, and it has also practical application in different forms including nanomicelles, NPs, microspheres, hydrogels, biofilms, and tablets (Rege, Garmise, & Block, 2003; Rezvani, Mohammadnejad, Narmani, & Bidaki, 2018). Furthermore, functional surface hydroxyl and amine groups facilitate their chemical modification to surface engineering in the CS backbone (Zhang, Ding, Yu, & Ping, 2007).

Also, CS as N-deacetylated derivative of chitin is a copolymer of glucosamine and N-acetyl-d-glucosamine. The micelle form of CS-based nanostructures (amphiphilic block copolymers) has shown broad spectrum of potency in anticancer agent delivery that are following by several research studies. In a work, Zhu, Cao, Cui, Qian, and Gu (2013) used the FA-functionalized N-succinyl-N′-octyl CS (FA-SOCS) micelle, as hydrophobic–hydrophilic core–shell, for targeted delivery system of 10-hydroxycamptothecin (HCPT) anticancer agent to targeted delivery. Average diameter size of these NPs was 122–145 nm. HCPT due to its insolubility in water, short half-life, and high toxicity to normal tissue cells needs to active delivery. Also, HCPT undergoes lactone ring-opening hydrolysis to inactive carboxylate form in physiological conditions and it is instable in body serum (Wang, Shang, Li, & Jiang 2009; Watanabe, Kawano, Toma, Hattori, & Maitani, 2008). Therefore, Zhu, Cao, Cui, Qian, and Gu (Zhu et al., 2013) applied the FA-targeted CS micelles to solve the water insolubility, provide sustained release and mainly improve the active delivery of this drug. Moreover, FITC-labeled FA-SOCS micelle remarkably evidenced the high internalization in in vitro and in vivo, while no HCPT signal was observed in free FA micelle uptake study. The outer hydrophilic layer of micelles is so crucial for improving the prolong circulation, escaping the immune clearance, and substantial accumulation in cancer site (Huo et al., 2010; Zhang, Huang, & Li, 2014). The pharmacokinetic behavior of the free HCPT, HCPT/SOC, and HCPT/folic acid-SOC micelles were investigated in the rats via I.V. administration. The total body clearance rates of free HCPT were about sevenfold faster than those HCPT/folic acid-SOC micelles, which have been shown translational development of targeted NPs in clinical applications. Also, plasma concentration rate of targeted micelles was more than four times in respect to nontargeted ones. On the other hand, tumor targeting potency of targeted micelles was investigated in Bel-7402 tumor-bearing mice by means of near infrared dye. As, targeted micelles were sharply detectable in the tumor site just 6 hr post-injection, it was 24 hr for nontargeted micelles. Moreover, it was revealed that NPs above 200 nm can be rapidly eliminated from the vascular pathway by macrophages and particles below 10 nm can be easily filtered by the kidney (Zhu et al., 2013).

In a research by Fathi et al. (2017), the maleic anhydride moieties were conjugated to hydroxyl sites of CS to produce the hydrophobic/hydrophilic nanomicelles. In order to this, sodium dodecyl sulfate/CS complex (SCCS) has been used for solubilizing CS in common organic solvents and dissolved in maleic anhydride to produce CS (O-maleoyl-SCCS) nanocomplex. Then, this organosoluble complex dissolved in N-isopropylacrylamide/oleic acid as hydrophilic/hydrophobic moieties to formation of N-isopropylacrylamide-oleic acid (PNIPAAm-co-OA)-g-CS micelles with free amino groups (mean particle size ∼100 nm). These intact amino groups are site of FA conjugation. Subsequently, erlotinib (ETB), as hydrophobic agent was physically encapsulated into micelles for treatment of metastatic advanced non-small cell lung and pancreatic cancers (Fathi et al., 2017). Eventually, it was concluded that the (Fluorescein isothiocyanate)-modified micelles can be delivered rapidly and effectively (more than six times internalization in comparison to nontargeted NPs) into the targeted cancer cells, significantly (Fathi et al., 2017). On the other hand, hemolysis assay was carried out to evaluate hemocompatibility of folic acid-modified micelles. As, the hemolytic ratio of the targeted micelles was less than 3%, that was in accordance with the guidelines of ISO/TR 7406 (Fathi et al., 2017).

In the amphiphilic polymers, hydrophobic core region serves as a reservoir for hydrophobic drugs, while hydrophobic core stabilization, polymers water-solubility of polymer is the fundamental role of hydrophilic shell (Adams, Lavasanifar, & Kwon, 2003). On the other hand, the various amphiphilic polymers can be formed micelles or self-aggregates nanocomplex in isotropic aqueous solution which is extended by intra- and intermolecular association between hydrophobic segments, primarily to minimize interfacial free energy (Mortensen, 2001). The rheological feature, small hydrodynamic radius, and thermodynamic stability are unique properties of polymeric micelles.

2.1.2 PLGA NPs and PLA NPs

Currently, the most important therapy approaches to circumvent side effects of chemotherapy and Multi drug resistance (MDR) are the use of biocompatible and biodegradable nano-biopolymers such as PLGA poly(lactide-co-glycolide), PLA (polylactic acid), and so forth in chemotherapeutic anticancer delivery (Figure 4; Hami et al., 2014; Kim et al., 2008). The PLGA is approved by FDA and it is classified safe for clinical use, as pharmaceutical excipients. Also, the great drug release behavior, excellent ability associate, and good ability of penetration the transmucosal are the main properties of PLGA NPs (Prabaharan et al., 2009; Zhou, Patel, Michael, Bertram, & Saltzman, 2012). On the other hand, the negative surface charge of PLGA makes it as suitable carrier in surface functionalization and drug conjugation and limits its interaction in normal cells.

In a research by (Wang, Zhao, Su, et al., 2010), the self-assemble cationic PLGA/FA which had been coated PEGlated polymeric liposome (PLS) core–shell NPs (FA-PLGA-PLS NPs) was developed to codelivery of pEGFP as a model of DNA and doxorubicin (DOX), as a model drug. So DOX was encapsulated and pEGFP was bound to the FA-PLGA-PLS NPs. In order to these syntheses, the cationic PEGlated amphiphilic octadecyl-quaternized lysine modified chitosan (PEG-OQLCS), cationic folic acid conjugated amphiphilic octadecyl-quarternized lysine modified chitosan (FA-OQLCS), and cholesterol (weight ratio 1/1/1, total lipids 30 mg) were dissolved in chloroform to create the organic phase. Cholesterol helps to maintain the structural stability of lipid shell. Then, chloroform was separated and thin film of lipid bilayer was obtained. Subsequently, the lipid film was dispersed in PLGA nanospheres solution and it was stirred to produce self-assemble micelles with maximum particle size ∼100 nm. The roles of PEG-OQLCS as protective coat and FA-OQLCS as targeted part are promoting the in vivo stability and monodispersity, and enhancing tumor accumulation and reducing the systemic toxicity, respectively (Wang, Zhao, Su, et al., 2010). Drug release (more than 50%) time of this NP was happened at first 6 hr. Codelivery efficiency of PLGA/FPL NPs was by 46.6%, while it was by 5.78 and 15.06% for free PLGA and FA-PLGA-PLS NPs, respectively, that it has been shown the FA-induced targeted delivery effects (Wang, Zhao, Su, et al., 2010). On the other hand, internalization efficiency was demonstrated about more than 50-fold for targeted micelles; as the bright red and green fluorescences were considerably observed in the cytoplasm around the nuclei which were indicated suitable codelivery of drug and gene. These results were demonstrated excellent potency of FA-based targeted therapy in clinical application.

In a report by Kuo and Chen (2015), the lactoferrin (Lf)- and FA-grafted PLGA NPs (Lf/FA/PLGA NPs; 181.9 ± 4.5 nm particle size with positive surface charge) were developed for targeted delivery of etoposide to inhibit the Glioblastoma (GBM) growth and penetrate in monolayer of human brain-microvascular endothelial cells (HBMECs). The Lf has been applied to permeate an in vitro blood–brain barrier (BBB) via receptor-mediated transcytosis (RMT) (Huang et al., 2007). Moreover, Lf can mediate the trigger the RMT pathway and accelerates the transportation of Lf/FA/PLGA NPs to HBMECs and makes a crucial role in delivering etoposide across the BBB by docking it. The sustained release was recorded for more than 50% of etoposide at first 7 days. On the other hand, the uptake of Lf/FA/PLGA NPs by U87MG cells was revealed the delivery of antitumor etoposide to brain cancer cells that was strongly related to FA (Kuo & Chen, 2015). Also, Lf could improve the uptake of Lf/FA/PLGA NPs by U87MG cells via binding to low-density lipoprotein receptor. Furthermore, the tiny NPs can be penetrate from BBB, however effective penetration was obtained via mediating specific blood proteins, including transferrin, leptin, insulin-like growth factors, and specifically Lf (Lf showed more effective penetration potency than others). Moreover, the other same work was carried out by (Xu, Jiang, Yu, et al., 2017) in order to active delivery of insulin in diabetic (as lethal disease) models. As specified, the subcutaneous needle injection of insulin is concomitant with lethal pain in patients. In order to overcome this disadvantage, FA-modified Ins-PLGA/CS NPs with 252.4 ± 4.6 nm in diameters were fabricated via electrostatic self-assembly approach by Xu et al. research work. The sustained release of insulin was about 35% at first 6 hr for globular positive-charge Ins-PLGA/FA-CS NPs. Furthermore, the serum which had been sustained release of insulin was significantly demonstrated that serum insulin level was maintained at 40 μIU/mL for long period time and subsequently it was decreased the percentage of initial blood glucose levels to half of maximum levels (100%; Xu, Jiang, Yu, et al., 2017). On the other hand, the enzyme inhibition test was performed to investigate the stability of insulin. Insulin is easy to damage in the presence of pepsin or pancreatin in gastrointestinal fluid. It was shown that about 10% of free insulin can be remained for 30 min in simulated gastric fluid after incubation and simulated intestinal fluid, while more than 80% of insulin can be remained after encapsulation of insulin into nanocarriers. Subsequently, many values of the insulin have been degraded for the free insulin by increasing the incubation time, while it was less than 40% for Ins-PLGA/FA-CS. Also, pharmacokinetics investigation of this micelle has been shown that oral delivery of insulin solution has no hypoglycemic effect, while blood glucose level has been reduced significantly after subcutaneous injection of 5 IU/kg insulin solution. Eventually, it was indicated that high hypoglycemic efficacy of the Ins-PLGA/FA-CS in the diabetic rats is related to the following features: the nanocomplex serves as an efficient tool for protection of insulin in acidify environment of the stomach, and the nanocomplex might increase the cellular permeability of the insulin and improve cellular uptake of Ins-PLGA/FA-CS nanocomplex (Xu, Jiang, Yu, et al., 2017).

In order to suppress the inappropriate activated macrophages, Mohammadi et al. (2016) were applied triblock copolymers FA-poly(l-lactide)-b-poly(ethylene glycol) (FA-PEG-PLLA), poly(l-lactide)-polyethylenimine-poly(l-lactide) (PLLA-PEI-PLLA), and PLLA-poly(ethylene glycol)-PLLA (PLLA-PEG-PLLA), as three-layered micelle to target FR-β that has high expression in this macrophages. FR-α has negligible accessibility to circulate folic acid-targeted drugs, because of its expression on the apical side of healthy tissues, while FR-β has great accessibility for activated macrophages. The three-layered micelle was optimized for the encapsulation of plasmid DNA at N/P ratio of 12. Subsequently, the transfection efficiency of micelle containing plasmid GFP-DNA was ascertained via isolation of spleen macrophages. The in vitro and in vivo FITC-labeled micelles high fluorescence intensity of FITC was quantified and a shift to higher fluorescence indicates the expression of FR in activated macrophages (more than 10 times in comparison to resting macrophages). Also, Green fluorescent protein (GFP) expression was shown more than three times to evaluate the transgene expression and results of gene expression in activated macrophages in comparison to resting macrophages. Gene release was determined with hydrogel retardation assay and no gene release was observed in physiologic pH condition, however about 15.3% DNA were released after 144 hr incubation in acidic pH (Mohammadi et al., 2016).

These amphiphilic micelles have excellent advantages such as good solubility of hydrophobic drugs, high loading capacity, great biocompatibility and biodegradability, reducing the nonspecific uptake by the reticuloendothelial system (RES), enhancing circulation time in blood, optimizing the anticancer efficiency of drug, overcoming the drug's drawbacks through minimizing its toxicity, overcoming the development of drug resistances, and siting specific targeting delivery (Hu et al., 2008; Ortiz et al., 2012; Xiao et al., 2012). In order to increase the drug's anti-angiogenic efficacy and enhance the antitumor efficacy, the curcumin-loaded copolymer FA-PEG-PLA, as amphiphilic micelles, were synthesized by (Yang, Chen, Zhao, et al., 2014). This nanocarrier was prepared by thin-film hydration method with mPEG2000-PLA2000 and Folate-PEG3000-PLA2000 (with 9:1 weight ratio) with average size of 70 nm. The drug loading and encapsulating efficiency of curcumin were by 4.84% ± 0.01% and 80.73% ± 0.16%. Moreover, the pharmacokinetic investigations in rats demonstrated that a three times enhancement in the half-life was achieved for Cur-loaded micelle formulations which were relative to solubilized Cur (Yang, Chen, Zhao, et al., 2014). Also, internalization studies were shown more than five times enhancement in cellular uptake.

2.1.3 Dextran NPs and hyaluronic acid NPs

Dextran (DEX), as biodegradable and biocompatible nanosize biomolecule, is a complex branched polysaccharide which has been made of many glucose molecules and has chains of varying lengths from 3 to 2,000 kD that straight chain consists of α-1,6 glycosidic linkages between glucose molecules (Figure 4). The saccharide-based nanocarriers are developed in order to improve the high anticancer efficacy, decrease the drug side effects, and prolong the drug circulation time in bloodstream, and so forth (Takara et al., 2012; Zhang et al., 2012). In a research by Hao, Ma, Huang, He, & Yao (2013), the bovine serum albumin-dextran-doxorubicin-FA (DOX/BSA-DEX-FA) conjugate was prepared by an esterification reaction between FA and dextran that fallowed by Maillard reaction between the DEX-FA and BSA to obtain BSA-DEX-FA with 90 nm in size. BSA has some advantages for NPs including reduced plasma protein adsorption on the particle surface, ease of NP purification, low cost, and unusual ligand-binding properties (Elzoghby, Samy, & Elgindy, 2012; Mohanta, Madras, & Patil, 2012). Also, BSA NPs were used in multilayer thin film via layer-by-layer self-assembly for targeting delivery. DOX loading amount and loading efficiency of the NPs are larger than 14 and 90%, respectively. Furthermore, drug release investigation was indicated more than fourfold sustained release versus free drug release. In this work, pH adjusting and heating process was applied to modulate the electrostatic and hydrophobic interactions between DOX and BSA, induce the self-aggregation of DOX and the denaturation/aggregation/gelation of BSA. On the other hand, the in vivo antitumor results were shown about three times survivability in DOX/BSA-DEX-FA treated animal models which were relative to nontargeted sample treatments (Hao et al., 2013). As, the tumor inhibition and survivability efficacies of DOX/BSA-DEX-FA nanocomplex were evaluated on H22 tumor-bearing mice. The results were shown that tumors inhibition rates (n = 5 per every group) of the DOX which has been loaded nanocomplex are nearly same in comparison to free DOX at the same dose of 5 mg/kg. However, the groups treated with the nanocomplex increase their body weights considerably, while the free DOX group decreases the body weight extremely. Also, increasing the dose of nanocomplex led to enhance the tumor inhibition rate, whereas it was toxic to mice body for free DOX. These results suggest that the toxicity of targeted nanocomplex is so lower than the toxicity of free DOX and even DOX/BSA-DEX nanocomplex. Hao et al. (2013) were also investigated the nanocomplexes which have been produced by heating for 30 and 70 min. The results indicate that the nanocomplexes produced by heating for 30 min have better antitumor activity in comparison to other ones that it can be due to longer heat treatment and decomposition of the FA during the heat treatment.

In the other DEX-based DDS by Zhao et al. (2017), the resveratrol (RSV) as polyphenol was developed to cancer growth inhibition with antioxidant and antimutagen aspects. RSV can inhibit the cancers via activating the mitochondrial apoptotic pathway with induces apoptosis to inhibit cancerous cell proliferation and improves the sensitivity of drug resistant cancer cells (Mattarei et al., 2013). FA-DEX-RSV (mean particle size ∼140 nm) as polymeric micelle, has several great properties, including better stability compared to surfactant micelles, enhanced solubilizing power, longer circulating time which is owing to outer hydrophilic shell, small size, and targeting capability. The free RSV demonstrated around ∼20% of apoptotic fractions while FA-DEX-RSV indicated a threefold higher apoptosis. Moreover, drug release of FA-DEX-RSV nanomicelle was observed by ∼40 and ∼30% after 24 hr at acidic and normal pHs. Also, the cellular internalization of FA-DEX-RSV was more than eightfold in comparison to free RSV (Zhao et al., 2017). It was revealed that RSV-DF is associated with the higher expression of p53, caspase-3, and BCL2 associated X gene, apoptosis regulator (BAX) than the free RSV and higher level of BAX and caspase-3 was further demonstrated the involvement of mitochondria-dependent apoptosis in the anticancer efficacy of FA-DEX-RSV micelle (Zhao et al., 2017).

In recent decade, hyaluronic acid (HA) has also been widely evaluated for use in tumor-targeted DDS due to its ability in specifically bind to various cancer cells (Jing, Zhang, Zhou, Liu, & Zhang, 2013). HA, as anionic glycosaminoglycan, is a main component of the extracellular matrix that has major roles in cell proliferation and migration. Also, HA is a biodegradable, biocompatible, nontoxic, nonimmunogenic, and bioavailable polysaccharide. This polysaccharide is a ligand of CD44 hyaluronan receptors, which are overexpressed in a variety of tumor types including prostate and breast cancers (Choi et al., 2012; Jin et al., 2012). Furthermore, the self-assembled small size in aqueous conditions and enhance the enhanced permeability and retention (EPR) effects are other properties of this nanosized polymeric micelles. In a study by Qiu et al. (2014), the FA and poly(l-histidine) conjugated HA (FA-HA-PHis) micelle, as pH-responsive amphiphilic copolymer was developed for acid-triggered rapid release of DOX in cancer cells. The results were revealed that, FA-HA-PHis micelles (154.8 ± 1.6 nm in diameter) can selectively uptake via CD44 receptor-mediated endocytosis and rapidly disassemble in early endosomes which are owing to pH-induced protonation of PHis and hyaluronidase action. However, after endocytosis inhibition experiments, it was observed that FA-HA-PHis micelles were mainly internalized via clathrin-mediated endocytosis and DOX was delivered to lysosomes in this conditions (Qiu et al., 2014). On the other hand, the effect of the macropinocytosis pathway on the uptake of micelles was evaluated using colchicine (macropinocytosis pathway inhibitor) and the results were indicated reduction in the internalization (reduce to 3%) of targeted nanocomplex that imply macropinocytosis has main role in targeted-cell uptake. Self-assembled hydrophobized polysaccharide polymeric micelles are significantly exhibited high water-solubility for hydrophobic drug carrier, excellent solubilization capacity and stability, sustained drug release, prolonged circulation, and tumor localization aspects (Lee, Lee, & Park, 2008). Moreover, Liu et al. (2011) were synthesized dual targeting paclitaxel-loaded FA-HA-C18 micelles (FA-HA-PTX; 191.9 ± 8.7 nm in diameter) with drug encapsulation efficiency of ∼97.3% and drug release by ∼55.2% at 192 hr. Substantially, five endocytic pathways were known for surplus values of micelle internalization: FA-mediated endocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and clathrin- and caveolae-independent endocytosis. These results were obtained by employing nanomolar dosages of several endocytic inhibitors such as hypertonic sucrose solution to dissociate of the clathrin, indometacin to inhibit the caveolae-mediated endocytosis, and chlorpromazine to dissociate the complex of clathrin and AP2 protein. However, FA- and clathrin-mediated endocytosis were considered as the most prominent mechanism for the micelle internalization (Liu et al., 2011).

2.2.1 Polyamidoamine NPs

Dendritic architecture is one of the most general topologies which has been observed in biological systems. Polyamidoamine (PAMAM) is one of the most studied dendrimers as the DDS (Figure 5). PAMAM NPs are nanosized (1–10 nm), three-dimensional, highly branched macromolecules which are consisting of three distinct components: a core, a hyperbranched mantle, and terminal functional groups (Narmani et al., 2017). These NPs are extremely soluble in water with many reactive end amine groups that make it as excellent nanocarrier for diagnostic and therapeutic goals (Narmani, Kamali, Amini, Salimi, & Panahi, 2018; Xie et al., 2014). In a research by Xu et al. (2016), FA and borneol (BO), well-known safe materials, which had been derived from traditional Chinese medicine, were applied for targeting delivery of DOX to glioma treatment. BO facilitates the BBB permeability and reduces the cytotoxic effects of PAMAM. On the other hand, the coupling BO on the surface of FA-PAMAM/DOX (167 nm in diameter) has several advantages for nanocomplex including increase its transportation from BBB (more than twofold), enhance its distribution agents in brain, prolong its circulation half-life time, improve DOX accumulation in brain tumor. Also, FA was improved the internalization efficiency until fourfold in cancer cell. Drug loading and entrapment efficiency of nanocomplexes were by 6.64% ± 0.09% and 64.58% ± 0.85%, and drug release of nanocomplex was by 28.2 and 48.8% over 24 hr at pH 7.4 and 5.5, respectively (Xu et al., 2016). For preclinical evaluation, the DOX plasma concentrations after intravenous injection of DOX, BO-PAMAM/DOX, and FA-BO-PAMAM/DOX were investigated via I.V. administration in rats (n = 5 per every group). As, DOX-loaded BO-PAMAM and FA-BO-PAMAM nanocomplexes were increased the area under the concentration-time curve (AUC0-inf) by 11.24- and 11.71-fold for BO-PAMAM/DOX and FA-BO-PAMAM/DOX, respectively. Furthermore, elimination half-life time (T1/2β) and mean residence time (MRT) of FA-BO-PAMAM were equal to 12.60 and 16.58 hr, while they were equal to 11.66 and 16.31 hr for BO-PAMAM, respectively. These results significantly support the extended residence time and control release profile of encapsulated drug in PAMAM as compared to free DOX in body (Xu et al., 2016). In another study, Narmani, Yavari, and Mohammadnejad (Narmani et al., 2017) were developed FA-PAMAM-PEG-5FU-^99mTc in order to imaging and therapeutic goals. In the in vivo study, their synthetic nanocomplex was effectively targeted breast cancer cells (fourfold better than nontargeted complex) in tumor bearing mice (n = 3 per every group). Moreover, their targeted nanocomplex could be accumulated in tumor site after 4 hr and tumor accompanied with liver were absorbed more than 75% of nanocomplex.

In the last decade, RNAs as anticancer agents have also been developed to target cell signaling intermediates to cancer cell inhibition. RNA-mediated therapeutic approaches can be selectively downregulated molecular targets in signaling pathways that are responsible for cell proliferation and significantly reduced multidrug resistance in comparison to drugs (Lo et al., 2010). Xu, Yeudall, & Yang, (2017) prepared FA-PAMAM NPs for active delivery of siRNA against vascular endothelial growth factor A (siVEGFA) in head and neck squamous cell carcinomas. The VEGFA is a main regulator of angiogenesis and promotes tumor development in tumor tissues, so that its knockdown has been shown to be effective in inhibiting tumor growth. FA-PAMAM-siVEGFA was exhibited FR-α mediated tumor uptake as excellent nanocomplex for local delivery of siRNAs and sustained retention properties. As it was considered, the very low systemic toxicity, sustained local drug release, high drug bioavailability, and excellent chemotherapeutic efficacy were advantages of this localized targeted delivery.

2.2.2 Polyethyleneimine NPs

Polyethyleneimines (PEIs) as cationic polymers have been applied as nucleic acids and drugs delivery carriers both in vitro and in vivo (Figure 5). Their surface positive charge, high drug loading, and good conjugation efficiency make them be able to combine with the therapeutic anticancer agents via electrostatic interactions (Benjaminsen, Mattebjerg, Henriksen, Moghimi, & Andresen, 2013). In a research by Wu, Zhang, Zhang, Sun, Wu, and Tang (2016), the PEG-b-(ɛCL-g-PEI)-b-ɛCL triblock copolymer (core–shell micelle) was applied for codelivery of P-glycoprotein (P-gp) siRNA and doxorubicin (DOX) to reverse MDR in breast cancer. The fabricated nanocomplex can effectively prevent renal clearance, RNase degradation and aggregation in circulation, minimize opsonization during circulation bloodstream, and protect micelles from mononuclear phagocytic system in circulation. After internalization, proton sponge effect of the branched PEI in the endosome help to endosomal elude of nanocomplex (Wu et al., 2016). The apoptosis levels of nanocomplex were measured by flow cytometric that they were obtained by 85.3, 41.3, and 15.7% for micelleplexes (PEG-DOX-b-(ɛCL-g-PEI)-b-ɛCL-siRNA), PEG-DOX-b-(ɛCL-g-PEI)-b-ɛCL, and free DOX, respectively. This high therapeutic efficiency of PEG-DOX-b-(ɛCL-g-PEI)-b-ɛCL-siRNA owes to overcome the drug efflux pump-mediated drug resistance. On the other hand, the histological analysis of tumor sections which has been stained with H&E was shown minimal residual cancer cells for micelleplexeses in comparison to free DOX. These results were indicated that micelleplexeses could considerably codeliver targeted nanocomplex to tumor tissues and subsequently produce more severe cancer cell apoptosis and tumor necrosis (Wu et al., 2016). Ultimately, the excellent internalization and effective tumor growth inhibition were observed in tumor bearing mice.

In other work by Shi et al. (2013), C60-PEI-FA nanocomplex was synthesized for targeting delivery of docetaxel (N-debenzoyl-N-tert-butoxy-carbonyl-10-deacetyl taxol, DTX) as a new class of hydrophobic taxane drugs with higher patient response rates and fewer side effects. DTX can inhibit the microtubule depolymerization to free tubulin in cancer cell. Fullerene (C60), as the third allotrope of carbon, is nanosize carbon material with unique photo-, electro-chemical, and physical properties which has excellent potency in delivery of hydrophobic therapeutic agents. This nanocomplex (140 ± 2.7 nm size in diameter) was indicated the prolonged blood circulation more than threefold tumor growth inhibition and apoptosis and 7.5-fold higher DTX uptake of tumor. Furthermore, the high tumor absorption through FR, which has been mediated internalization and EPR effect with low kidney clearance, was obtained in pharmacokinetic investigations (Shi et al., 2013). On the other hand, oligodeoxynucleotide delivery into cancer cells is a critical challenge in the cancer therapy. PEI NPs have been significantly developed to delivery of oligodeoxynucleotide vectors with high efficiency and low toxicity. In a study, Yang et al. (2015) were developed OA modified and FA decorated PEI NPs (FA-PEI-OA NPs) for delivery of LOR-2501 as antisense oligonucleotide against ribonucleotide reductase R1 subunit. OA could significantly improve the delivery efficacy of oligodeoxynucleotide. FA-PEI-OA-LOR-2501 nanocomplex was remarkably induced the downregulation of R1 mRNA and R1 protein. The higher cellular uptake was observed with PEI-OA/LOR-2501 at N/P ratio of 6. After inhibition of clathrin-mediated uptake in cancer cells, the clathrin-mediated uptake process was inhibited by 80.5% that demonstrated the clathrin-mediated internalization of nanocomplex (Yang et al., 2015). Also, the same results were obtained in cytosine deaminase/5-fluorocytosine (CD/5-FC) and TNF-related apoptosis-inducing ligand (TRAIL) genes delivery to glioma cells and rats via FA-PEG-PEI nanocomplex (N/P ratio of 15) that was reported by Liang et al. (2009). This research group was developed combined therapy of CD/5-FC with TRAIL genes against rat C6 glioma models. According to this study, average tumor size for the Phosphate-buffered saline (PBS)-control groups was equal to 172.52 ± 8.02 mm³, while 53.13 ± 3.72 mm³ noted for the combined therapy against glioma tumor (Liang et al., 2009). The cross-sensitization between TRAIL gene and 5-FC chemotherapeutic in this nanocomplex could induce apoptotic pathway through caspase activation and might be an effective therapeutic strategy for C6 gliomas.