1. Polymersomes as Drug Carrier

The loading of hydrophilic substrates into polymersomes can either be passive or active. A passive loading strategy involves adding the drug to both the aqueous phase and the inner water phase of the polymersomes ^[1][39]. This approach involves the diffusion of uncharged drug molecules across the outer structure to reach the core of vesicles. The low or high core pH accounts for the substrate to become charged. With this method, it is possible to achieve high encapsulation efficiency and stable retention of the drug molecules. In remote loading, polymersomes with thicker membranes may prove advantageous since they enable the substrate to diffuse rapidly ^[2][40]. Köthe et al. developed a novel method called DAC (Dual asymmetric centrifugation) to prepare polymersomes, where poly caprolactone and polyethylene glycol block copolymers were used as precursor molecules. The particle prepared showed high efficiency in drug loading of both hydrophobic and hydrophilic moieties. The method used no organic solvents and did not involve any post-processing steps, making it a facile process for the fabrication of polymersomes as the best suitable drug carrier ^[3][41]. Polymersomes can be encapsulated and decorated with targeting ligands by combining small and large molecules with a block copolymer. It may be possible to extend the release time of drugs when they are incorporated into vesicles by using biodegradable linkers that improve the retention of the drug. Walvekar et al. studied the potential role of polymersomes in the delivery of antibiotics against MRSA (Methicillin-resistant Staphylococcus aureus) infections. The particle was synthesized using a self-assembly technique, encapsulated with vancomycin drug, and conjugated with oleylamine- hyaluronic acid ligands. The in vitro study results indicated that this particle would act as a promising drug carrier for the treatment of bacterial infections ^[4][42]. Another study by Porges et al. also developed polymersomes for the treatment against antibacterial infections. Block copolymer-based polymersomes were fabricated and encapsulated with antibiotics for the treatment of Burkholderia infections. It was concluded that the developed polymersomes would serve as the best carrier for antibiotics ^[5][43]. A transmembrane channel protein can be incorporated into a polymersome membrane to increase its permeability for hydrophilic molecules and ions. It has been shown, for example, that in cascade reactions enzymes encapsulated in membranes containing channel proteins can avoid inhibiting factors and also maintain the ability to access the substrate. This has been demonstrated in the synthesis of polymersomes with cytidine-monophosphate-N-acetylneuraminic acid and OmpF G119D (channel protein) to retain access to the substrate. Alibolandi et al. developed dextran based polymersomes for insulin delivery. The particle was prepared using the hydration method and encapsulated with insulin. The in vivo and in vitro study results demonstrated that the particle loaded with insulin significantly delivered the payload via an oral drug delivery system ^[6][44]. As part of more fundamental studies on membrane proteins, polymersome membranes had the ability to be tuned in their mechanical properties by adjusting the chemical composition, allowing for a better insertion of membrane proteins. The functionalization of transmembrane proteins is limited by the thickness of the membrane since most of the channel proteins are designed for only lipid membranes. PMOXA-b-PDMS-b-PMOXA model demonstrates that polymersome membrane conformations adapt to allow biopores smaller than hydrophobic membrane layers ^[7][8][45,46]. When polymersomes are made from long block copolymers, their membranes can have a thickness of up to 50 nanometers. Accordingly, compared to liposomes, polymersomes are theoretically capable of accommodating greater amounts of hydrophobic molecules ^[9][47]. Furthermore, thick membranes can lead to a slower release of hydrophobic substrates because the diffusional distances are much higher. When the vesicle is formed, the hydrophobic molecules or drugs occupy the organic part and incorporate it into the membrane. A 47% encapsulation efficiency was achieved by polymersomes made up of poly(trimethylene carbonate) and poly(L-glutamic acid) loaded with doxorubicin by nanoprecipitation at pH 10.5 ^[10][48]. A low diffusion distance may result in low drug retention when loading substrates. There is a risk of a high rate of drug release in polymersomes due to the narrow diffusion distance, despite their membrane thickness. A biodegradable polymer might further impair the release profile of the cargo in vivo, as a high polymer degradation rate could result in the destabilization of the membrane and an acceleration of the cargo release process ^[11][49].

2. Polymersome-Based Targeted Anticancer Drug Delivery

It has been over a decade since multiple chemotherapeutics were developed for the treatment of cancer, but in spite of this there are still many challenges in the field of chemotherapeutics like less selectivity, physiological barriers, half-life, and poor bioavailability ^[12][60]. Additionally, due to the damage to the liver and kidney caused by cancer therapeutics, chemotherapy may even be discontinued in the course of treatment. Moreover, resistance to anticancer drugs is a major concern because it occurs due to abnormal expression of drug-efflux transporters, which prevents the intracellular transport of cancer therapeutics. This is because drug carriers can improve the bioavailability of anticancer drugs, enhance permeability and retention (EPR) effect, reduce side effects, and provide efficient delivery of chemotherapeutics bypassing the biological barriers ^[13][61]. Since polymersome-based drug delivery systems are characterized by a stable structure like a cell with a membrane and lumen that are size-controlled, they have been investigated as smart cancer therapeutics nanocarrier systems ^[14][62]. It is possible for polymersomes to encapsulate both hydrophobic and hydrophilic anticancer agents in their lumens or membranes. Furthermore, the polymers can be functionalized with ligands complementary to receptors on the target site as well as with ligands that are sensitive to environmental responsiveness, so that they can deliver drugs precisely and release them in response to stimuli ^[15][63]. Polymersomes offer an excellent opportunity to target postsurgical premetastatic niches and microresiduals, resolving the challenges of poor therapeutic delivery ^[16][64]. Several studies have demonstrated the synergistic antitumor effect of combination chemotherapy and immunotherapy for cancer. After proper combination with chemotherapy, immunotherapy can significantly improve response rates and efficacy. A study by Japir et al. suggests that tumor-dilatable polymersome nanofactories that are capable of long-term intratumoral retention may represent a promising strategy to enhance enzyme prodrug chemo-immunotherapy using polymersomes ^[17][65]. As a result of targeted delivery systems, the target markers in cancer cells that are overexpressed or unique are targeted ^[18][66]. A number of cancer targets have been identified, and a number of targeting ligands have been employed in the development of polymersome-based targeted delivery systems, including folate, antibodies, etc. ^[19][67]. For example, Polymersomes containing folate are able to target cancer cells by means of endocytosis through the folate receptor. In order to survive a complicated physiological environment and provide efficient targeting capability, these targeted ligands have been conjugated primarily with polymersomes by covalent bonding ^[20][21][68,69]. The folate molecule contains two carboxylic acid groups, which greatly influence polymersome reactivity. In cancer, antibodies may bind specifically to antigens that are over-expressed or those that are cancer-specific. A number of studies had been carried out in the early stages to achieve targeted therapy by conjugating antibodies onto anticancer drugs. Lee et al. conducted in vitro study of anticancer drug delivery to a specific site of breast cancer cells by conjugation of antibodies. Diblock co-polymeric substances containing polyethylene glycol and poly (lactic co-glycolic acid) were synthesized using the solvent evaporation technique. During this fabrication, indocyanine green and doxorubicin were encapsulated in the polymer molecules. Since breast cancer cells overexpress HER2 gene, a complementary binding agent-anti-HER2 antibody was conjugated through the carbodiimide crosslinking method. The experimental results showed that the cell death rate was much higher in the polymersomes encapsulated with the dual drug compared with a single drug dosage. Hence this study indicates that drugs encapsulated in polymeric structure readily reduced the chemotoxicity and increased the therapeutic effect ^[22][70]. Clinical trials or even marketing approvals have already begun for several antibody-drug conjugates for cancer treatment. However, there is a low ratio of drug to antibody in the antibody-drug conjugate, which means excessive antibodies are consumed when preparing antibody-drug conjugate, resulting in reduced drug-to-antibody ratios. Alternative options include conjugating antibodies to polymersomes encapsulated with drugs ^[23][24][71,72]. The development of polymersome-based targeted delivery systems has currently been accomplished using several types of antibodies, including anti-EGFR antibodies (cetuximab, trastuzumab), anti-CD44 antibodies, anti-PSCA antibodies, and anti-EpCAM antibodies, among others. The monoclonal antibody AM6 was used to target breast cancer cells. Khanna et al. recently reported that the nanocarrier prepared using poly (lactic co-glycolic acid) encapsulated with paclitaxel drug showed better targeting to breast cancer cells due to the surface functionalization of AM6 antibody. The antibody was conjugated using the reaction chemistry between thiol and maleimide. The results obtained from in vitro and in vivo determine that the antibody-conjugated particle actively targeted perlecan in cancer cells and significantly helped to facilitate the drug release ^[25][73]. In cancer cells, short peptides of specific sequences bind to abnormally overexpressed proteins (integrin or transferrin) membrane and enhance their ability to penetrate the cells’ membranes. These peptides were found to have significant abilities to target cancer cells and could be conjugated with polymersomes to provide their ability to target. WREAAYQRFL (tumor-homing peptide) was used by Oz et al. to design an integrin-targeted polymersome ^[26][74]. This study showed that the peptide was capable of recognizing and binding to αvβ3 integrin, which is pivotal in tumor growth, spread, and metastasis. Breast cancer cells typically overexpress this integrin. Thiol-ene click chemistry was used to chemically link the targeting peptide to a diblock copolymer. The peptide-conjugated polymers were then assembled to form polymersomes to encapsulate the anticancer drug doxorubicin during the self-assembly process. The use of tetraiodothyroacetic acid, triphenylphosphonium cation, and biotin has also been considered for targeted delivery solutions based on polymersomes ^[27][75]. By amination between the amino group and p-anisic acid on polymer chains, anisamide can be incorporated into polymersomes for its affinity to overexpressed sigma receptors in the tumor region. It is also important to note that these polymersome-based targeted delivery systems have the potential to target not only the overexpressed receptors found at tumor sites but also some tissue constituents, like the divalent calcium ions found in hydroxyapatite that accounts for bones formation, which can serve as potential targeting element. Since it was found that folate receptors are abundant in the gastrointestinal tract, oral delivery of anticancer agents is now the best choice of treatment. Hence, Pan et al. worked on the fabrication of polymersome-based carriers suitable for oral drug delivery systems. The polymersomes were prepared with polylactic acid and pluronic F127 and decorated with folic acid ligands. The particle was loaded with paclitaxel and was added to the particle to improve the absorption of the drug Vitamin E TPGS. The in vitro and in vivo results proved that the particle exhibits higher cellular toxicity compared with free drugs. The bioavailability was readily increased due to the specific interaction between the folate receptor and folic acid ligand. Consequently, this particle can be effective in the case of oral delivery ^[28][59]. The physiological barriers such as the blood–brain barrier (BBB) and blood–tumor barrier (BTB) remain as major obstacles to the treatment of brain tumors, including gliomas. In order to overcome both the BBB and the BTB, a dual-targeted or multitargeted polymersome delivery system is highly regarded. Research conducted by Figueiredo et al. concluded that overexpression of protein-1 related to low-density lipoprotein in BBB could serve as a target for efficient delivery. The polymersomes were developed with diblock copolymers encapsulated with doxorubicin and conjugated with angiopep-2. The angiopep-2 can actively target the over-expressed protein in BBB. The in vitro study results carried out on glioblastoma cells showed higher cytotoxic effects compared to free drugs. Thus, polymersomes conjugated with angiopep-2 can provide sustained release of the drug at its targeted site ^[29][76]. It was discovered by Chen et al. that an efficient polymersome delivery system attaching des-octanoyl ghrelin and folic acid effectively delivers the anticancer drug to brain tumors through dual-targeted polymersomes. The protein des-octanoyl ghrelin travels in the direction of blood to the brain, which may enhance drug-encapsulated polymersome transport across the BBB ^[30][77]. Folic acid could directly bind to folate receptors on cancer tissues after traversing the BBB to facilitate receptor-mediated endocytosis for drug concentration enhancement in the cancer cells. Polymersome-based delivery may improve delivery efficacy over conventional delivery systems, but still, there is a requirement to promote rapid drug release within tumor sites to improve therapeutic efficacy. In this regard, stimuli-responsive smart polymersomes can be more effectively used as a means of drug delivery system with controlled release profiles in response to stimulation ^[31][78].

3. Smart Polymersomes-Based Delivery Systems

There are many advantages to the intelligent delivery system, such as the delivery of the drugs effectively with rapid release at the tumor site reducing the probability of drug release in the bloodstream or surrounding healthy tissues ^[32][33][17,79]. The microenvironment of tumor tissues differs quite a bit from that of normal tissues, including pH, hypoxia, and overexpression of enzymes ^[34][35][36][80,81,82]. To construct polymersome-based smart delivery systems, it is necessary to take advantage of the specificity of the microenvironment in tumor tissues to design and build environment-responsive polymers. The response to external stimuli, such as ultrasound, light, or magnetic fields, can also be used. The smart polymersome-based delivery is classified into three categories depending on their source of stimulation and function. The chemical stimuli include the pH of the microenvironment, redox, reactive oxygen species, and ions, whereas the physical stimuli include temperature, heat, light, magnetic field, and ultrasound. Ultrasound waves will help to improve the release of the drug from the carrier into the target cell efficiently. Zhong et al. fabricated nanobubble carrier system using poly (lactic co-glycolic acid). The carrier was loaded with paclitaxel drug via double emulsion and conjugated with herceptin antibody via carbodiimide coupling reaction. The in vitro and in vivo study experiments showed that the herceptin decorated nanobubble reacted well to the ultrasound and promoted the targeting and release. This study concluded that this particle, when combined with ultrasound, could function as theranostic agent in image-guided therapy ^[37][83]. Other than these two, there are some biological stimuli like proteins, enzymes, and bioactive molecules like ATP. It has been reported that polymersomes that are pH- and redox-sensitive are most commonly found. Changes in pH can affect the structure or properties of pH reactive polymersomes. The introduction of pH-responsive groups is one way to achieve pH-responsive ionizability. Under alternant change in pH, the polymers can reversibly modify their pH-responsive properties, which can influence the accelerated release of the drug encapsulated within ^[38][84]. In addition to this approach, it is also possible to attach pH-sensitive bonds into polymer, after which the release of drugs in polymer chains can be accomplished by cleavage in chemical bonds or by pH-induced degradation of main chains and side chains. Therefore, polymersomes are stable at physiological pH; hence the structure or properties of polymersome can be altered to trigger the release of drugs in the pH of the microenvironment, which is lowered (acidic conditions). Albuquerque et al. developed block copolymer encapsulated with doxorubicin via microfluidic technology. This method produced monodispersed polymer and was further characterized using hi-end photophysical tools. The in vitro results showed that doxorubicin-encapsulated polymer possessed pH responsiveness towards tumor microenvironment and delivered the drug precisely ^[39][85]. For cells to proliferate and function properly, glutathione plays a critical role, and due to the rapid proliferation of cancer cells glutathione concentrations are at least four times higher in cancer cells than in those of healthy tissues ^[40][86]. During exposure to a reductive condition like a high glutathione environment, the disulfide bond cleaves to produce free thiol. In order to trigger glutathione-induced drug release, redox-responsive polymersomes were developed ^[41][87]. Therefore, through disulfide bonds, anticancer drugs can be covalently conjugated to amphiphilic polymers. Upon delivery to cancer cells, glutathione cleaves the disulfide bonds to release the anticancer drugs to the cancer cells. As a result of this method, researchwers are able to produce polymersomes containing anticancer drugs that have a high drug loading capacity to avoid the release of drugs in circulation. In addition, another approach is to design amphiphilic polymers that are composed of disulfide bonds along their main chains or on their side chains ^[42][88]. The cleavage of disulfide bonds in an environment with high glutathione concentrations produces alterations in hydrophobicity or dismantling of polymer structures, which results in the rapid release of payload as a result of the cleavage of disulfide bonds. Additionally, external physical stimuli can be precisely applied to specific tumor sites to minimize unnecessary side effects ^[43][89]. As a result of temperature-sensitive polymersomes, the chemical properties and morphology respond to it and release the drug rapidly. Polymersomes that respond to temperature have been developed using a variety of temperature-sensitive polymers ^[44][90]. As a noninvasive stimulus, ultrasound and magnetic fields have also been considered as promising possibilities. Polymersomes that respond to biological stimuli have also been developed using enzymes and ATP. Taking advantage of the microenvironment, the particle can be designed in a way to respond to the condition. The hypoxic surrounding is one of the common characteristics possessed by tumor cells that will contribute to the growth and relapse of tumors. This condition also limits the entry of drug carriers into solid tumor tissues. Designing a carrier, which could respond to hypoxia, will facilitate the entry and delivery. According to this hypothesis, Kulkarni et al. designed an echogenic particle that can respond to hypoxic stimuli. Polymersomes were fabricated with polyethylene glycol and poly lactic acid. The particle was then functionalized with azobenzene and peptide- iRGD for hypoxia responsiveness and tissue penetration, respectively, also encapsulating gemcitabine for treating pancreatic adenocarcinoma. The in vitro and in vivo results demonstrated that the polymersomes penetrated well into the solid tumor and delivered the drug efficiently. The echogenicity of the particle helped in imaging the study model under the ultrasound technique. Thus, it is concluded that the polymersomes designed above served as drug carriers and monitored the delivery simultaneously ^[45][50].