Chimaeric Polymersomes Research Articles

Bioresponsive Chimaeric Polymersomes Mediate Sustained and Liver-Specific siRNA Transfection In Vivo.

The silencing of disease-causing genes with small interfering RNA (siRNA) offers a particularly effective therapeutic strategy for different disorders; however, its clinical efficacy relies on the development of nontoxic and tissue-specific delivery vehicles. Herein, we report that bioresponsive chimaeric polymersomes (BCP) with short poly(ethylenimine) as inner shell mediate highly efficacious, sustained, and liver-specific siRNA transfection in vivo. BCP exhibited remarkable encapsulation efficiencies of siRNA (95-100%) at siRNA-feeding contents of 15-25 wt %, to afford stable, small-sized (55-64 nm), and neutral-charged BCP-siRNA. siApoB-Loaded BCP (BCP-siApoB) outperformed lipofectamine counterparts and silenced 93% of ApoB mRNA in HepG2 cells at 50 nM siApoB without inducing cytotoxicity. Intriguingly, the in vivo studies using wild-type C57BL/6 mice revealed that BCP-siApoB preferentially accumulated in the liver, and a single dose of 4.5 mg/kg achieved over 90% downregulation of ApoB mRNA for at least 10 days. The systemic administration of BCP-siApoB at 4.5 mg/kg every 2 weeks or 1.5 mg/kg weekly in diet-induced obese mice could also achieve up to 80% silencing of ApoB mRNA. The liver specificity and silencing efficacy of BCP-siApoB could further be improved by decorating it with the trivalent N-acetylgalactosamine (TriGalNAc) ligand. These bioresponsive and liver-specific chimaeric polymersomes provide an enabling technology for siRNA therapy of various liver-related diseases.

Targeted nanodelivery of siRNA against KRAS G12D inhibits pancreatic cancer.

Pancreatic cancer (PC) stands as a most deadly malignancy due to few effective treatments in the clinics. KRAS G12D mutation is a major driver for most PC cases, and silencing of KRAS G12D is considered as a potential therapeutic strategy for PC, which is nevertheless crippled by lacking a pragmatic delivery system for siRNA against KRAS G12D (siKRAS). Here, we report that cRGD peptide-modified bioresponsive chimaeric polymersomes (cRGD-BCP) mediate highly efficient siKRAS delivery to PANC-1 tumor, potently silencing KRAS G12D mRNA in tumor cells and effectively suppressing PC tumor growth in mice. cRGD-BCP exhibited remarkable encapsulation of siKRAS (loading content > 14 wt.%, loading efficiency > 90%) to form stable and uniform (ca. 68 nm) nanovesicles (cRGD-BCP-siKRAS). Of note, cRGD density greatly impacted the cellular uptake and silencing efficiency of cRGD-BCP-siKRAS in PANC-1 cells, in which an optimal cRGD density of 15.7 mol.% achieved 3.7- and 3.6-fold enhancement of internalization and gene silencing, respectively, compared with non-targeted BCP-siKRAS. cRGD-BCP-siKRAS was practically intact after 3-week storage at 4°C. Intriguingly, cRGD-BCP-siKRAS markedly enhanced the uptake of siKRAS in PANC-1 tumor, and at a siKRAS dose of 3 mg/kg knocked down 90% KRAS G12D gene, resulting in potent tumor inhibition and extraordinary survival benefits (median survival time: 101 days versus 38 (PBS group) and 59 days (BCP-siKRAS)) with 40% mice achieved complete regression. It appears that cRGD-mediated nanodelivery of siKRAS provides a potential cure for pancreatic cancer. STATEMENT OF SIGNIFICANCE: Small interfering RNA (siRNA) emerges as a specific and powerful biopharmaceuticals against cancers; however, inefficient in vivo delivery impedes its clinical translation. In spite of the fact that KRAS G12D mutation has been identified as a major driver for most pancreatic cancer, its notorious non-druggability renders little success on development of molecular targeted drugs. Pancreatic cancer is deemed as current king-of-cancer. Here, we show that cyclic RGD peptide installed bioresponsive polymersomes are able to efficiently deliver siRNA against KRAS G12D to pancreatic tumor, resulting in 90% gene knock-down and effective tumor inhibition. Strikingly, two out of five mice have been cured. This targeted nanodelivery of siRNA provides a high-efficacy treatment strategy for pancreatic cancer.

Polymersome-mediated cytosolic delivery of cyclic dinucleotide STING agonist enhances tumor immunotherapy

Cyclic dinucleotides (CDNs) as stimulator of interferon genes (STING) agonists capable of inducing strong antitumor innate immune response are highly promising for tumor immunotherapy. The efficacy of these CDNs is, however, reduced greatly by their fast clearance, poor cell uptake and inefficient cytosolic transportation. Here, we report that reduction-responsive biodegradable chimaeric polymersomes (CPs) markedly enhance tumor retention and cytosolic delivery of a synthetic CDN, ADU-S100, and bolster STING pathway activation in the tumor microenvironment and tumor draining lymph nodes, giving significantly better tumor repression and survival of B16F10 melanoma-bearing mice compared with free CDN control. The superiority of CPs-mediated CDN delivery is further verified in combination therapy with low-dose fractionated radiation, which brings about clearly stronger and longer-term immunotherapeutic effects and protection against tumor re-challenge. The development of nano-STING agonists that are able to overcome the delivery barriers of CDNs represents an effective strategy to potentiate cancer immunotherapy.

EGFR-targeted pemetrexed therapy of malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare malignancy with poor prognosis, for which chemotherapy with pemetrexed (PEM) is among the few clinical treatments. PEM suffers, however, fast clearance, moderate drug exposure, and dose-limiting toxicities. Here, we report on epidermal growth factor receptor (EGFR)-targeted disulfide-crosslinked biodegradable chimaeric polymersomes (EGFR-CPs) to firmly load PEM and boost chemotherapy of MPM. EGFR-CPs encapsulating 8.7-16.4 wt.% PEM (EGFR-CPs-PEM) showed diameters of 62-65nm and reduction-responsive drug release property. EGFR-CPs-PEM was more efficiently taken up by EGFR-overexpressed MSTO-211H cells, inducing about 4.7-fold enhanced anticancer activity compared with non-targeted CPs-PEM control. Intriguingly, the in vivo experiments in MSTO-211H xenograft mouse model revealed that EGFR-CPs-PEM brought about superior tumor deposition and penetration to CPs-PEM, and significantly more potent tumor repression than CPs-PEM and free PEM. This polymersome-enabled EGFR-targeted delivery of PEM offers an appealing therapeutic strategy for MPM.

Selective transferrin coating as a facile strategy to fabricate BBB-permeable and targeted vesicles for potent RNAi therapy of brain metastatic breast cancer in vivo

Brain metastases are a most disturbing situation for breast cancer patients as there is basically no adequate treatment available. Any potential drug formulation has to be able to cross the blood-brain barrier (BBB) and specific to metastatic brain tumors without causing unacceptable adverse effects. Here, we developed transferrin-functionalized chimeric polymersomes carrying siRNA against polo-like kinase 1 (Tf@TBP-CPs-siPLK1) for treating brain metastatic MDA-MB 231 triple negative breast cancer (TNBC) xenografts in mice. To facilitate the loading of siPLK1, chimaeric polymersomes (CPs) were designed with spermine in the watery core and transferrin-binding peptide (TBP) at the surface, enabling attachment of transferrin after the siRNA loading step and thereby circumventing interference of transferrin with siRNA loading. Tf@TBP-CPs-siPLK1 encapsulating 3.8 wt% siRNA had a mean size of about 50 nm and a neutral zeta potential in phosphate buffer (PB). By virtue of the presence of transferrin, Tf@TBP-CPs demonstrated greatly (ca. 5-fold) enhanced internalization in MDA-MB 231 cells and transcytosis in the endothelial (bEnd.3) monolayer model in vitro as well as markedly improved accumulation in the orthotopically xenografted MDA-MB 231 tumor in the brain in vivo compared with control CPs lacking transferrin, supporting that transferrin mediates efficient BBB penetration and high specificity towards MDA-MB 231 cells. As a result, Tf@TBP-CPs-siPLK1 effectively inhibited tumor progression and prolonged the lifespan of the mice significantly. Selective transferrin coating appears to be a particularly facile strategy to fabricate BBB-permeable and targeted vesicles for potent RNAi therapy of brain metastatic breast cancer.

Systemic administration of polymersomal oncolytic peptide LTX-315 combining with CpG adjuvant and anti-PD-1 antibody boosts immunotherapy of melanoma

Oncolytic peptide LTX-315 while showing clinical promise in treating solid tumors is limited to intratumoral administration, which is not applicable for inaccessible or metastatic tumors. The cationic and amphipathic nature of oncolytic peptides engenders formidable challenges to developing systems for their systemic delivery. Here, we describe cRGD-functionalized chimaeric polymersomes (cRGD-CPs) as a robust systemic delivery vehicle for LTX-315, which in combination with CpG adjuvant and anti-PD-1 boost immunotherapy of malignant B16F10 melanoma in mice. cRGD-CPs containing 14.9 wt% LTX-315 (cRGD-CPs-L) exhibited a size of 53 nm, excellent serum stability, and strong and selective killing of B16F10 cells (versus L929 fibroblasts) in vitro, which provoked similar immunogenic effects to free LTX-315 as revealed by release of danger-associated molecular pattern molecules. The systemic administration of cRGD-CPs-L gave a notable tumor accumulation of 4.8% ID/g and significant retardation of tumor growth. More interestingly, the treatment of B16F10 tumor-bearing mice was further boosted by co-administration of polymersomal CpG and anti-PD-1 antibody, in which two out of seven mice were cured as a result of strong immune response and long-term immune memory protection. The immunotherapeutic effect was evidenced by secretion of IL-6, IFN-γ and TNF-α, tumor infiltration of CD8+ CTLs and Th, and induction of TEM and TCM in spleen. This study opens a new avenue to oncolytic peptides, which enables durable immunotherapy of tumors via systemic administration.

Systemic Delivery of NAC-1 siRNA by Neuropilin-Targeted Polymersomes Sensitizes Antiangiogenic Therapy of Metastatic Triple-Negative Breast Cancer.

Antiangiogenic therapy with bevacizumab while being interesting for metastatic triple-negative breast cancer (mTNBC) is restrained by tumor hypoxia elevation and cancer stem cell enrichment. Here, we find that neuropilin-1 (NRP-1)-targeted delivery of nucleus accumbens-associated protein-1 (NAC-1) siRNA mediated by tLyP-1 peptide-functionalized chimaeric polymersomes (tLyP-1-Ps) effectively sensitizes antiangiogenic therapy of mTNBC in vivo. tLyP-1-Ps showed good encapsulation (up to 14.4 wt. %) of siNAC-1, giving robust tLyP-1-Ps-siNAC-1 nanoformulation with a defined size of 48.5 nm (PDI = 0.13) and a surface charge of -9.2 mV, and mediated efficient cytoplasmic transportation of siNAC-1 in MDA-MB-231 TNBC cells, resulting in significant silencing of NAC-1 mRNA and the corresponding oncoprotein. Transwell invasion and wound healing assays revealed that tLyP-1-Ps-siNAC-1 potently inhibited MDA-MB-231 cell invasion and migration. Intriguingly, tLyP-1-Ps-siNAC-1 was shown to markedly improve the bevacizumab therapy of mTNBC, significantly curbing lung metastasis and prolonging the survival time of the MDA-MB-231 metastatic model. The combination of targeted NAC-1 gene silencing and antiangiogenic therapy appears to be an innovative treatment for mTNBC.

GE11 peptide-installed chimaeric polymersomes tailor-made for high-efficiency EGFR-targeted protein therapy of orthotopic hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains a leading malignancy with a high mortality and little improvement in treatments. Protein drugs though known for their extraordinary potency and specificity have rarely been investigated for HCC therapy owing to lack of appropriate delivery systems. Here, we designed GE11 peptide-installed chimaeric polymersomes (GE11-CPs) for high-efficiency EGFR-targeted protein therapy of orthotopic SMMC-7721 HCC-bearing nude mice. GE11-CPs were assembled from poly(ethylene glycol)-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate)-b-poly(aspartic acid) (PEG-P(TMC-DTC)-PAsp) and GE11-functionalized PEG-P(TMC-DTC), which allowed efficient loading and protection of proteins in the watery interior and fine-tuning of GE11 densities at the surface. CPs with short PAsp segments (degree of polymerization (DP) = 5, 10 and 15) exhibited a protein loading efficiency of 60%-72% and glutathione-responsive protein release. Saporin-loaded GE11-CPs had a size of 36 - 62 nm depending on GE11 densities and DP of PAsp. Notably, GE11-CPs with 10% GE11 revealed greatly enhanced uptake in SMMC-7721 cells, boosting the anticancer potency of saporin for over 3-folds compared with non-targeted control (half-maximal inhibitory concentration (IC50) = 11.0 versus 36.3 nM). The biodistribution studies using Cy5-labeled cytochrome C as a model protein demonstrated about 3-fold higher accumulation of GE11-CPs formulation than CPs counterpart in both subcutaneous and orthotopic SMMC-7721 tumor models. Notably, saporin-loaded GE11-CPs revealed low toxicity, effective tumor inhibition and significant improvement of survival rate compared with PBS and non-targeted groups (median survival time: 99 versus 37 and 42 days). EGFR-targeted chimaeric polymersomes carrying proteins appear an interesting HCC treatment modality.

CD44-targeted vesicles encapsulating granzyme B as artificial killer cells for potent inhibition of human multiple myeloma in mice

Multiple myeloma (MM) is a malignant blood cancer homing in bone marrow that is particularly hard to treat. The effective treatment for MM shall be not only MM-selective but also capable of homing to bone marrow. Herein, we report on hyaluronic acid-directed reduction-responsive chimaeric polymersomes encapsulating a key player in the NK cells, granzyme B (HA-RCP-GrB) as an artificial killer cell for targeted protein therapy of MM. Interestingly, HA-RCP-GrB displayed high MM-targetability and anti-MM activity with a remarkably low IC50 of 8.1 nM toward CD44 overexpressing LP1 human MM cells. The in vivo biodistribution studies using Cy5-labeled cytochrome C as a model protein demonstrated significantly enhanced accumulation of HA-RCP in the subcutaneous LP1 tumor as well as in the bone marrow of orthotopic LP1 MM model compared with the non-targeted RCP counterparts, confirming that HA-RCP possesses MM-selectivity and is able to deliver proteins to the bone marrow. In accordance, HA-RCP-GrB exerted significantly better suppression of subcutaneous LP1 tumor than the non-targeted RCP-GrB. More interestingly, in the orthotopic LP1 MM-bearing mice, HA-RCP-GrB led to significant survival benefits and less body weight loss over PBS and RCP-GrB. μCT analyses, H&E and TRAP staining revealed that mice treated with HA-RCP-GrB had greatly reduced osteolysis and proliferation of atypical plasma cells in the bone marrow. HA-RCP-GrB has emerged as a novel and effective treatment for multiple myeloma.

Oncoprotein Inhibitor Rigosertib Loaded in ApoE-Targeted Smart Polymersomes Reveals High Safety and Potency against Human Glioblastoma in Mice

The unbiased cytotoxicity and blood-brain barrier (BBB) impermeability render common chemotherapeutics nonviable for treating glioblastoma (GBM) patients. Although rigosertib (RGS), a RAS effector protein inhibitor, has shown low toxicity to healthy cells and high efficacy toward various cancer cells by inactivating PI3K-Akt, it hardly overcomes the BBB barricade. Here, we report that RGS loaded in apolipoprotein E derived peptide (ApoE)-targeted chimaeric polymersomes (ApoE-CP) is safe and highly potent against human GBM in vivo. ApoE-CP exhibited stable loading of RGS in its lumen, giving RGS nanoformulations (ApoE-CP-RGS) with a size of 60 nm and reduction-triggered drug release behavior. Notably, ApoE-CP-RGS induction markedly enhanced the G2/M cell cycle arrest and inhibitory effect in U-87 MG glioblastoma cells compared with the nontargeted CP-RGS and free RGS. The therapeutic outcomes in orthotopic U-87 MG GBM models demonstrated that ApoE-CP-RGS brought about effective GBM inhibition, greatly prolonged survival time, and depleted adverse effects. Rigosertib formulated in ApoE-targeted chimaeric polymersomes has emerged as a novel, highly specific, efficacious, and nontoxic treatment for glioblastoma.

Boosting RNAi therapy for orthotopic glioblastoma with nontoxic brain-targeting chimaeric polymersomes

Glioblastoma with intracranial infiltrative growth remains an incurable disease mainly owing to existence of blood brain barrier (BBB) and off-target drug toxicity. RNA interference (RNAi) with a high specificity and low toxicity emerges as a new treatment modality for glioblastoma. The clinical application of RNAi technology is, however, hampered by the absence of safe and brain-targeting transfection agents. Here, we report on angiopep-2 peptide-decorated chimaeric polymersomes (ANG-CP) as a nontoxic and brain-targeting non-viral vector to boost the RNAi therapy for human glioblastoma in vivo. ANG-CP shows excellent packaging and protection of anti-PLK1 siRNA (siPLK1) in its lumen while quickly releasing payloads in a cytoplasmic reductive environment. Notably, in vitro experiments demonstrate that ANG-CP can effectively permeate the bEnd.3 monolayer, transport siRNA into the cytosol of U-87 MG glioblastoma cells via the LRP-1-mediated pathway, and significantly silence PLK1 mRNA and corresponding oncoprotein in U-87 MG cells. ANG-CP greatly prolongs the siPLK1 circulation time and enhances its accumulation in glioblastoma. RNAi with siPLK1 induces a strong anti-glioblastoma effect and significantly improves the survival time of glioblastoma carrying mice.

Lung cancer specific and reduction-responsive chimaeric polymersomes for highly efficient loading of pemetrexed and targeted suppression of lung tumor in vivo

Lung cancer is one of the worldwide leading and fast-growing malignancies. Pemetrexed disodium (PEM, Alimta®), a small hydrophilic drug, is currently used for treating lung cancer patients. However, PEM suffers from issues like fast elimination, low bioavailability, poor tumor cell selectivity and penetration. Here, we report on lung cancer specific CSNIDARAC (CC9) peptide-functionalized reduction-responsive chimaeric polymersomes (CC9-RCPs) for efficient encapsulation and targeted delivery of PEM to H460 human lung cancer cells in vitro and in vivo. PEM-loaded CC9-RCPs (PEM-CC9-RCPs) was obtained from co-self-assembly of poly(ethylene glycol)-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate)-b-polyethylenimine (PEG-P(TMC-DTC)-PEI) and CC9-functionalized PEG-P(TMC-DTC) in the presence of PEM followed by self-crosslinking. PEM-CC9-RCPs displayed an optimal CC9 density of 9.0% in targeting H460 cells, a high PEM loading content of 14.2 wt%, a small hydrodynamic size of ca. 60 nm and glutathione-triggered PEM release. MTT assays showed that PEM-CC9-RCPs was 2.6- and 10- fold more potent to H460 cells than the non-targeting PEM-RCPs and free PEM controls, respectively. Interestingly, PEM-CC9-RCPs exhibited 22-fold longer circulation time and 9.1-fold higher accumulation in H460 tumor than clinical formulation Alimta®. Moreover, CC9-RCPs showed obviously better tumor penetration than RCPs. Remarkably, PEM-CC9-RCPs at 12.5 mg PEM equiv./kg effectively suppressed growth of H460 xenografts and significantly prolonged mouse survival time as compared to PEM-RCPs and Alimta® controls. These lung cancer specific and reduction-responsive chimaeric polymersomes provide a unique pemetrexed nanoformulation for targeted lung cancer therapy. Statement of SignificanceMultitargeted antifolate agent pemetrexed (PEM, Alimta®) is currently used for treating lung cancer patients and has low side-effects. However, PEM suffers from issues like fast elimination, low bioavailability, poor tumor cell selectivity and penetration. Scarce work on targeted delivery of PEM has been reported, partly because most conventional nanocarriers show a low and instable loading for hydrophilic, negatively charged drugs like PEM. Herewith, we report on lung cancer specific CSNIDARAC (CC9) peptide-functionalized reduction-responsive chimaeric polymersomes (CC9-RCPs) which showed efficient PEM encapsulation (14.2 wt%, 60 nm) and targeted delivery of PEM to H460 human lung cancer cells, leading to effective suppression of H460 tumor xenografts and significantly prolonged survival rates of mice than Alimta®. To the best of our knowledge, this represents a first report on targeted nanosystems that are capable of efficient loading and targeted delivery of PEM to lung tumors.

Virus-Mimicking Chimaeric Polymersomes Boost Targeted Cancer siRNA Therapy In Vivo.

Small interfering RNA (siRNA) offers a highly selective and effective pharmaceutical for various life-threatening diseases, including cancers. The clinical translation of siRNA is, however, challenged by its short plasma life, poor cell uptake, and cumbersome intracellular trafficking. Here, cNGQGEQc peptide-functionalized reversibly crosslinked chimaeric polymersomes (cNGQ/RCCPs) is shown to mediate high-efficiency targeted delivery of Polo-like kinase1 specific siRNA (siPLK1) to orthotopic human lung cancer in nude mice. Strikingly, siRNA is completely and tightly loaded into the aqueous lumen of the polymersomes at an unprecedentedly low N/P ratio of 0.45. cNGQ/RCCPs loaded with firefly luciferase specific siRNA (siGL3) or siPLK1 are efficiently taken up by α3 β1 -integrin-overexpressing A549 lung cancer cells and quickly release the payloads to the cytoplasm, inducing highly potent and sequence-specific gene silencing in vitro. The in vivo studies using nude mice bearing orthotopic A549 human lung tumors reveal that siPLK1-loaded cNGQ/RCCPs boost long circulation, superb tumor accumulation and selectivity, effective suppression of tumor growth, and significantly improved survival time. These virus-mimicking chimaeric polymersomes provide a robust and potent platform for targeted cancer siRNA therapy.

Efficacious delivery of protein drugs to prostate cancer cells by PSMA-targeted pH-responsive chimaeric polymersomes

Protein drugs as one of the most potent biotherapeutics have a tremendous potential in cancer therapy. Their application is, nevertheless, restricted by absence of efficacious, biocompatible, and cancer-targeting nanosystems. In this paper, we report that 2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid (Acupa)-decorated pH-responsive chimaeric polymersomes (Acupa-CPs) efficiently deliver therapeutic proteins into prostate cancer cells. Acupa-CPs had a unimodal distribution with average sizes ranging from 157–175nm depending on amounts of Acupa. They displayed highly efficient loading of both model proteins, bovine serum albumin (BSA) and cytochrome C (CC), affording high protein loading contents of 9.1–24.5wt.%. The in vitro release results showed that protein release was markedly accelerated at mildly acidic pH due to the hydrolysis of acetal bonds in the vesicular membrane. CLSM and MTT studies demonstrated that CC-loaded Acupa10-CPs mediated efficient delivery of protein drugs into PSMA positive LNCaP cells leading to pronounced antitumor effect, in contrast to their non-targeting counterparts and free CC. Remarkably, granzyme B (GrB)-loaded Acupa10-CPs caused effective apoptosis of LNCaP cells with a low half-maximal inhibitory concentration (IC50) of 1.6nM. Flow cytometry and CLSM studies using MitoCapture™ revealed obvious depletion of mitochondria membrane potential in LNCaP cells treated with GrB-loaded Acupa10-CPs. The preliminary in vivo experiments showed that Acupa-CPs had a long circulation time with an elimination phase half-life of 3.3h in nude mice. PSMA-targeted, pH-responsive, and chimaeric polymersomes have appeared as efficient protein nanocarriers for targeted prostate cancer therapy.

Chimaeric polymersomes based on poly(ethylene glycol)-b-poly(l-leucine)-b-poly(l-glutamic acid) for efficient delivery of doxorubicin hydrochloride into drug-resistant cancer cells

Chimaeric polymersomes based on poly(ethylene glycol)-b-poly(l-leucine)-b-poly(l-glutamic acid) for efficient delivery of doxorubicin hydrochloride into drug-resistant cancer cells

Anisamide-Decorated pH-Sensitive Degradable Chimaeric Polymersomes Mediate Potent and Targeted Protein Delivery to Lung Cancer Cells.

In spite of their high potency and specificity, few protein drugs have advanced to the clinical settings due to lack of safe and efficient delivery vehicles. Here, novel anisamide-decorated pH-sensitive degradable chimaeric polymersomes (Anis-CPs) were designed, prepared, and investigated for efficient and targeted delivery of apoptotic protein, granzyme B (GrB), to lung cancer cells. Anis-CPs were readily prepared with varying Anis surface densities from anisamide end-capped poly(ethylene glycol)-b-poly(2,4,6- trimethoxybenzylidene-1,1,1-tris(hydroxymethyl)ethane methacrylate)-b-poly(acrylic acid) (Anis-PEG-PTTMA-PAA) and PEG-PTTMA-PAA copolymers. Using cytochrome C (CC) as a model protein, Anis-CPs displayed high protein loading efficiencies (40.5-100%) and loading contents (up to 16.8 wt %). CC-loaded Anis-CPs had narrow distribution (PDI 0.04-0.13) and small sizes ranging from 152 to 171 nm, which increased with increasing CC contents. Notably, the release of proteins from Anis-CPs was accelerated under mildly acidic conditions, due to the hydrolysis of acetal bonds in PTTMA. MTT assays showed that GrB-loaded Anis-CPs (GrB-Anis-CPs) displayed high targetability to sigma receptor overexpressing cancer cells such as H460 and PC-3 cells. For example, GrB-Anis-CPs exhibited increasing antitumor efficacy to H460 cells with increasing Anis contents from 0 to 80%. The antitumor activity of GrB-Anis-CPs was significantly reduced upon pretreating H460 cells with haloperidol (a competitive antagonist). Notably, the half-maximal inhibitory concentrations (IC50) of GrB-Anis70-CPs were determined to be 6.25 and 5.94 nM for H460 and PC-3 cells, respectively, which were 2-3 orders of magnitude lower than that of chemotherapeutic drugs, such as paclitaxel. Flow cytometry studies demonstrated that GrB-Anis70-CPs induced widespread apoptosis of H460 cells. The confocal laser scanning microscopy (CLSM) experiments using FITC-labeled CC-loaded Anis-CPs confirmed fast internalization and intracellular protein release into H460 cells. GrB-Anis-CPs with high potency and specificity are particularly interesting for targeted therapy of lung cancers.

Galactose-Decorated Reduction-Sensitive Degradable Chimaeric Polymersomes as a Multifunctional Nanocarrier To Efficiently Chaperone Apoptotic Proteins into Hepatoma Cells

Hepatoma-targeting reduction-sensitive chimaeric biodegradable polymersomes were designed and developed based on galactose-poly(ethylene glycol)-poly(ε-caprolactone) (Gal-PEG-PCL), PEG-PCL-poly(2-(diethylamino)ethyl methacrylate) (PEG-PCL-PDEA, asymmetric), and PEG-SS-PCL for facile loading and triggered intracellular delivery of proteins. The chimaeric polymersomes formed from PEG-PCL-PDEA and PEG-SS-PCL had a monodisperse distribution with average sizes ranging from 95.5 to 199.2 nm depending on PEG-SS-PCL contents. Notably, these polymersomes displayed decent loading of bovine serum albumin (BSA), ovalbumin (OVA), and cytochrome C (CC) proteins likely due to presence of electrostatic and hydrogen bonding interactions between proteins and PDEA block located in the interior of polymersomes. The in vitro release studies showed that protein release was largely accelerated under a reductive condition containing 10 mM dithiothreitol (DTT). For example, ca. 77.2 and 22.1% of FITC-BSA were released from CP(SS50) (chimaeric polymersomes containing 50 wt % PEG-SS-PCL) at 37 °C in 12 h in the presence and absence of 10 mM DTT, respectively. Confocal microscopy showed that FITC-CC-loaded Gal-decorated CP(SS40) could efficiently deliver and release FITC-CC into HepG2 cells following 24 h treatment, in contrast to little or negligible fluorescence detected in HepG2 cells treated with FITC-CC-loaded nontargeting polymersomes or free CC. MTT assays revealed that CC-loaded Gal-decorated CP(SS40) exhibited apparent targetability and pronounced antitumor activity to HepG2 cells, in which cell viabilities decreased from 81.9, 60.6, 49.5, 42.2 to 31.5% with increasing Gal-PEG-PCL contents from 0, 10, 20, 30 to 40 wt %. Most remarkably, granzyme B-loaded Gal-decorated chimaeric polymersomes effectively caused apoptosis of HepG2 cells with a markedly low half-maximal inhibitory concentration (IC(50)) of 2.7 nM. These reduction-responsive chimaeric biodegradable polymersomes offer a multifunctional platform for efficient intracellular protein delivery.

PH-sensitive degradable chimaeric polymersomes for the intracellular release of doxorubicin hydrochloride

pH-sensitive degradable chimaeric polymersomes were developed based on asymmetric poly(ethylene glycol)-b-poly(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl)ethane methacrylate)-b-poly(acrylic acid) (PEG-PTTMA-PAA) triblock copolymers for active loading as well as triggered intracellular release of hydrophilic doxorubicin hydrochloride (DOX·HCl). PEG-PTTMA-PAA copolymers were readily prepared with Mn PAA ranging from 1.5, 2.1 to 2.7 kg/mol by sequential reversible addition-fragmentation chain transfer (RAFT) copolymerization of 2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl)ethane methacrylate (TTMA) and acrylic acid (AA) using PEG-CPADN (Mn PEG = 5.0 kg/mol; CPADN: 4-cyanopentanoic acid dithionaphthalenoate) as a macro-RAFT agent. PEG-PTTMA-PAA copolymers formed mono-disperse polymersomes with average sizes of 63.9–112.1 nm, which decreased with increasing Mn PAA. The polymersomal structure was confirmed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Notably, the acetals in polymersomes while sufficiently stable at pH 7.4 were prone to rapid hydrolysis at mildly acidic pHs of 4.0 and 5.0, which resulted in swelling and eventually disassembly of polymersomes. These chimaeric polymersomes could actively load DOX·HCl resulting in remarkably high drug loading contents (up to 15.9 wt.%) and loading efficiencies (up to 88.8%). The in vitro release studies showed that DOX·HCl was released from chimaeric polymersomes in a controlled and pH-dependent manner. CLSM observations revealed that these chimaeric polymersomes could efficiently deliver and release DOX·HCl into the nuclei of HeLa cells. MTT assays in HeLa cells demonstrated that DOX·HCl-loaded PEG-PTTMA-PAA polymersomes exhibited high anti-tumor activity with IC50 (inhibitory concentration to produce 50% cell death) of 1.48–1.67 μg/mL, close to that of free DOX·HCl, while blank polymersomes were practically non-toxic up to a tested concentration of 2.0 mg/mL. These pH-sensitive degradable chimaeric polymersomes have appeared to be a promising alternative to liposomes for tumor-targeted delivery of DOX·HCl.

Biodegradable chimaeric polymersomes mediate highly efficient delivery of exogenous proteins into cells

Biodegradable chimaeric polymersomes based on asymmetric poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(2-(diethylamino) ethyl methacrylate) (PEG-PCL-PDEA) triblock copolymers were designed for encapsulation and delivery of exogenous proteins into cells. These chimaeric polymersomes showed remarkably high protein loading efficiencies (up to 100%) and loading contents (up to 56.4 wt.%) for various types of proteins. Notably, these polymersomes could escape from the endosomes and deliver proteins into the cytoplasm. Furthermore, they could simultaneously transport proteins and doxorubicin into the cytoplasm and cell Nucleus, respectively.

Polymersomes Spanning from Nano- to Microscales: Advanced Vehicles for Controlled Drug Delivery and Robust Vesicles for Virus and Cell Mimicking

Polymersomes provide a highly promising platform for mimicking biological membranes as well as for controlled delivery of various pharmaceuticals and biopharmaceuticals. The recent advancement has made it possible to prepare small to giant polymersomes spanning from nano- to microscales, stimuli-sensitive polymersomes that swell or collapse in response to an external or internal stimulus, chimaeric polymersomes that contain distinct interior environments separated from the outside by an asymmetric membrane, porous polymersomes with tailored permeability, biomimetic and targeting polymersomes that selectively deliver drugs, proteins, and/or imaging probes to the sites of action. In this Perspective, we will give a brief introduction to polymersomes, highlight recent design and preparation of several sophisticated polymersomes, and discuss future challenges.