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Transferrin receptor (TfR) is a promising target validated in the clinical trials for managing various malignancies. Transferrin (Tf) and single chain antibody fragment can target TfR and are typically conjugated to nanomedicines via post-modification, which poses significant production challenges. Here, we report that the polymersomes functionalized with a Tf-binding peptide CGGGHKYLRW (TBP-Ps) can selectively and stably bind Tf and subsequently mediate targeted doxorubicin (Dox) delivery to TfR over-expressing HCT-116 colorectal cancer cells in vitro and in vivo. The Tf surface density of the polymersomes could be controlled by the surface content of TBP. Interestingly, modifying Dox-loaded TBP-Ps with Tf led to greatly increased cellular uptake and inhibitory effect of HCT-116 cells. Tf-bound TBP-Ps demonstrated rapid accumulation in the tumor xenografts in nude mice following i.v. injection. More importantly, Dox-loaded Ps with Tf binding significantly enhanced the antitumor efficacy in mice bearing HCT-116 tumors compared to polymersomes without Tf binding. Surface functionalization of the nanoparticles with Tf-binding peptide provides an appealing strategy in formulating Tf-targeted nanomedicines.

The clinical use of protein therapeutics with intracellular targets is hampered by its in vivo fragility and low cell permeability. Here, we report that cell-selective penetrating and reduction-responsive polymersomes (CPRPs) mediate high-efficiency targeted delivery of granzyme B (GrB) to orthotopic human lung tumor in vivo. Model protein studies using FITC-labeled cytochrome C (FITC-CC) revealed efficient and high protein loading up to 17.2 wt% for CPRPs. FITC-CC-loaded CPRPs exhibited a small size of 82-90 nm, reduction-responsive protein release, as well as greatly enhanced internalization and cytoplasmic protein release in A549 lung cancer cells compared with the non-targeted FITC-CC-loaded RPs control. GrB-loaded CPRPs showed a high potency toward A549 lung cancer cells with a half maximal inhibitory concentration (IC50) of 20.7 nM. Under the same condition, free GrB was essentially non-toxic. Importantly, installing cell-selective penetrating peptide did not alter the circulation time but did enhance tumor accumulation of RPs. Orthotopic A549-Luc lung tumor-bearing nude mice administered with GrB-loaded CPRPs at a dosage of 2.88 nmol GrB equiv./kg showed complete tumor growth inhibition with little body weight loss throughout the treatment period, resulting in significantly improved survival rate over the non-targeted and non-treated controls. These cell-selective penetrating and reduction-responsive polymersomes provide a targeted protein therapy for cancers.

There is tremendous interest in integrating CT imaging with chemotherapy; however, reported iodine-based nanosystems such as nanogels and nano-emulsions display typically reduced contrast coefficient, low drug loading and stability, and poor targetability. Here, cRGD-functionalized disulfide-crosslinked iodine-rich polymersomes (cRGD-XIPs) were designed as a novel, robust and smart theranostic agent and investigated for targeted CT imaging and chemotherapy of malignant tumors. Methods: cRGD-XIPs were prepared from co-self-assembly of poly(ethylene glycol)-b-poly(dithiolane trimethylene carbonate-co-iodinated trimethylene carbonate) (PEG-P(DTC-IC)) and cRGD-PEG-P(DTC-IC) block copolymers. In vitro and in vivo CT contrast effect of cRGD-XIPs was studied using αvβ3-overexpressing B16 melanoma as a tumor model in comparison with clinical agent iohexol. The therapeutic efficacy of doxorubicin-loaded cRGD-XIPs (cRGD-XIPs-Dox) to B16 melanoma was investigated and compared with XIPs-Dox (non-targeted), cRGD-IPs-Dox (non-crosslinked) and free Dox. Results: cRGD-XIPs were formed with 55.5 wt.% iodine and ca. 90 nm in diameter. cRGD-XIPs-Dox with a Dox loading of 15.3 wt.% bared superior colloidal stability and reduction-responsive drug release. Notably, blank cRGD-XIPs showed a maximum-tolerated dose (MTD) > 400 mg iodine equiv./kg while cRGD-XIPs-Dox had an MTD > 150 mg Dox equiv./kg, ca. 15-fold improvement over free Dox. cRGD-XIPs revealed superior CT contrast effect and achieved 46.5- and 24.0-fold better enhancement of CT imaging of B16 melanoma than iohexol at 4 h following intratumoral and intravenous injection, respectively. cRGD-XIPs-Dox displayed an elimination half-life of 6.5 h and an elevated accumulation of 6.68% ID/g in the tumors. Furthermore, cRGD-XIPs-Dox was significantly more effective than XIPs-Dox and cRGD-XPs-Dox in inhibiting growth of B16 melanoma model. Conclusion: This proof-of-concept study demonstrates that cRGD-XIPs are a robust, non-toxic and smart polymeric theranostic agent that can not only significantly enhance CT imaging of tumors but also mediate efficient tumor-targeted chemotherapy. XIPs offer a unique and safe platform for theranostic polymersomes that pre-select patients using CT imaging prior to targeted chemotherapy with the same system.

Biodegradable nanopolymersomes with an ultrahigh iodine content for high-performance CT imaging in vivo were designed. To obtain iodine-rich nanopolymersomes, we devised and prepared a new iodine-functionalized trimethylene carbonate (IC) monomer in two straight steps with an overall yield of 25% by the Finkelstein reaction. Gel permeation chromatography displayed a unimodal distribution with a moderate Mw/Mn of 1.40, confirming successful synthesis of PEG-b-PIC. Interestingly, PEG-b-PIC had an ultrahigh iodine content of 60.4 wt%, which is significantly higher than iohexol and all reported iodinated nanoparticle platforms. Remarkably, the PEG-b-PIC diblock copolymer was readily self-assembled into stable nanopolymersomes. Dynamic light scattering (DLS) showed a small hydrodynamic size of =100 nm with a low polydispersity index =0.10. Transmission electron microscopy (TEM) image confirmed their small size and vesicle structure, likely due to their thick membrane and the inherent PEG stealth ability. The stability of iodine conjugated with an aliphatic chain is usually weaker than that conjugated with aromatic derivatives. However, unlike traditional small molecules, all iodine atoms in IPs are sequestered in the hydrophobic membrane of polymersomes, which would effectively protect iodine from leakage. The in vivo SPECT/CT images showed that in sharp contrast to fast accumulation of Na125I in the thyroid and bladder, I-labeled IPs had a long circulation time and predominantly accumulated in the RES like spleen and liver, further corroborating that IPs are stable in circulation and iodine leakage is negligible.