Cell surface patching via CXCR4-targeted nanothreads for cancer metastasis inhibition.

The binding of therapeutic antagonists to their receptors often fail to translate into adequate manipulation of downstream pathways. To fix this 'bug', here we report a strategy that stitches cell surface 'patches' to promote receptor clustering, thereby synchronizing subsequent mechano-transduction. The "patches" are sewn with two interactable nanothreads. In sequence, Nanothread-1 strings together adjacent receptors while presenting decoy receptors. Nanothread-2 then targets these decoys multivalently, intertwining with Nanothread-1 into a coiled-coil supramolecular network. This stepwise actuation clusters an extensive vicinity of receptors, integrating mechano-transduction to disrupt signal transmission. When applied to antagonize chemokine receptors CXCR4 expressed in metastatic breast cancer of female mice, this strategy elicits and consolidates multiple events, including interception of metastatic cascade, reversal of immunosuppression, and potentiation of photodynamic immunotherapy, reducing the metastatic burden. Collectively, our work provides a generalizable tool to spatially rearrange cell-surface receptors to improve therapeutic outcomes.

Allogeneic "Zombie Cell" as Off-The-Shelf Vaccine for Postsurgical Cancer Immunotherapy.

Allogeneic tumor cell vaccines provide off-the-shelf convenience but lack patient specificity due to heterogeneity in tumor antigens. Here, allogeneic tumor cell corpses are converted into "zombie cells" capable of assimilating heterogeneous tumor by seizing cancer cells and spreading adjuvant infection. This causes pseudo-oncolysis of tumors, transforming them into immunogenic targets for enhanced phagocytosis. It is shown that in postoperative tumor models, localized delivery of premade "zombie cells" through stepwise gelation in resection cavity consolidates tumor surgery. Compared to analogous vaccines lacking "seizing" or "assimilating" capability, "zombie cell" platform effectively mobilizes T cell response against residual tumors, and establishes immunological memory against tumor re-challenge, showing less susceptibility to immune evasion. Despite using allogeneic sources, "zombie cell" platform functions as generalizable framework to produce long-term antitumor immunity in different tumor models, showing comparable effect to autologous vaccine. Together, with the potential of off-the-shelf availability and personalized relevance to heterogenous tumor antigens, this study suggests an alternative strategy for timely therapy after tumor surgery.

A Less-is-More Strategy for Mitochondria-Targeted Photodynamic Therapy of Rheumatoid Arthritis.

Conventional photodynamic therapy (PDT) of rheumatoid arthritis (RA) faces a dilemma: low-power is insufficient to kill pro-inflammatory cells while high-power exacerbates inflammation. Herein, mitochondrial targeting is introduced in PDT of RA to implement a "less-is-more" strategy, where higher apoptosis in pro-inflammatory cells are achieved with lower laser power. In arthritic rats, chlorine 6-loaded and mitochondria-targeting liposomes (Ce6@M-Lip) passively accumulated in inflamed joints, entered pro-inflammatory macrophages, and actively localized to mitochondria, leading to enhanced mitochondrial dysfunction under laser irradiation. By effectively disrupting mitochondria, pro-inflammatory macrophages are more susceptible to PDT, resulting in increased apoptosis initiation. Additionally, it identifies that high-power irradiation caused cell rupture and release of endogenous danger signals that recruited and activated additional macrophages. In contrast, under low-power irradiation, mitochondria-targeting Ce6@M-Lip not only prevented inflammation but also reduced pro-inflammatory macrophage infiltration and pro-inflammatory cytokine secretion. Overall, targeting mitochondria reconciled therapeutic efficacy and inflammation, thus enabling efficacious yet inflammation-sparing PDT for RA. This highlights the promise of mitochondrial targeting to resolve the dilemma between anti-inflammatory efficacy and inflammatory exacerbation in PDT by implementing a "less-is-more" strategy.

Immunogenic hydrogel toolkit disturbing residual tumor "seeds" and pre-metastatic "soil" for inhibition of postoperative tumor recurrence and metastasis.

Tumor recurrence and metastasis is the leading cause of mortality for postoperative breast cancer patients. However, chemotherapy intervention after surgery is often unsatisfactory, because residual microtumors are difficult to target and require frequent administration. Here, an all-in-one and once-for-all drug depot based on in situ-formed hydrogel was applied to fit the irregular surgical trauma, and enable direct contact with residual tumors and sustained drug release. Our immunological analysis after resection of orthotopic breast tumor revealed that postsurgical activation of CXCR4-CXCL12 signal exacerbated the immunosuppression and correlated with adaptive upregulation of PD-L1 in recurrent tumors. Thus, a multifunctional hydrogel toolkit was developed integrating strategies of CXCR4 inhibition, immunogenicity activation and PD-L1 blockade. Our results showed that the hydrogel toolkit not only exerted local effect on inhibiting residual tumor cell "seeds" but also resulted in abscopal effect on disturbing pre-metastatic "soil". Furthermore, vaccine-like effect and durable antitumor memory were generated, which resisted a secondary tumor rechallenge in 100% cured mice. Strikingly, one single dose of such modality was able to eradicate recurrent tumors, completely prevent pulmonary metastasis and minimize off-target toxicity, thus providing an effective option for postoperative intervention.

A liposome-based combination strategy using doxorubicin and a PI3K inhibitor efficiently inhibits pre-metastatic initiation by acting on both tumor cells and tumor-associated macrophages.

Pre-metastatic initiation is essential in tumor metastasis, and the inhibition of it could prevent the spread of cancers to distant organs. Both tumor-associated macrophages (TAMs) and the epithelial-mesenchymal transition (EMT) play an important role in the pre-metastatic initiation stage. Herein, a liposome-based combination strategy which involves doxorubicin-loaded liposomes (Lip-Dox) and PI3K inhibitor-loaded liposomes (Lip-LY) was developed to simultaneously regulate tumor cells and TAMs for inhibiting pre-metastatic initiation. In tumor cells, Lip-LY sensitized cells to Lip-Dox treatment and inhibited the EMT process which was promoted by succinate, further mitigating succinate-induced migration and invasion of 4T1 cells. In TAMs, Lip-LY could efficiently inhibit the polarization of TAMs and reduce the percentage of M2 TAMs, so as to exhibit synergistic effects with Lip-Dox in TAM-induced metastasis. As a result, the combination treatment successfully reduced the lung metastasis of 4T1 bearing BALB/c mice by destroying metastatic tumor cells and inhibiting pre-metastatic initiation with decreased metastasis-associated protein expression. Overall, our work provided a simple and promising combination strategy for inhibiting pre-metastatic initiation in multiple ways to treat cancer metastasis.

Trauma-Responsive Scaffold Synchronizing Oncolysis Immunization and Inflammation Alleviation for Post-Operative Suppression of Cancer Metastasis.

Tumor surgery can create an inflammatory trauma to aggravate residual tumor "seed" to colonize pre-metastatic niches (PMNs) "soil" at secondary sites, thereby promoting post-operative metastasis. However, two-pronged strategies for post-surgical elimination of asynchronous "seeds" and "soil" at different regions are currently lacking. Here, we have designed a hydrogel that can be injected into a resection cavity, where it immediately forms a scaffold and gradually degrades responding to enriched reactive oxygen species at adjacent trauma for local delivery and on-demand release of autologous cancer cells succumbing to oncolysis (ACCO) and anti-inflammatory agent. The autologous cell source self-provides a whole array of tumor-associated antigens, and the oncolysis orchestration of a subcellular cascade confers a self-adjuvanting property, together guaranteeing high immunogenicity of the ACCO vaccine that enables specific antitumor immunization. In parallel, inflammation alleviation exerted bidirectional functions to reshape the local immune landscape and resuscitate ACCO, leading to the eradication of residual tumor "seeds" while simultaneously intercepting the "seed-soil" crosstalk to normalize distant lung leading to regression of pre-existing PMN "soil". As a result, regional and metastatic recurrence were completely thwarted. Together, this framework synchronizing oncolysis immunization and inflammation alleviation provides an effective option for post-operative suppression of metastasis.

Concurrent impairment of nucleus and mitochondria for synergistic inhibition of cancer metastasis.

Cancer metastasis, which increases the mortality in a short period of time, has been considered as the main challenge in tumor treatment. However, tumor growth suppression also should not be ignored in cancer metastasis treatment. Recently, accumulating evidences have suggested that mitochondria play an important role in mitigating caner metastasis. Nucleus, as the repository of genetic information, plays a key role in cell proliferation. However, it remains elusive that the concurrent impairment of nucleus and mitochondria may achieve better anti-tumor and anti-metastatic effects. Here, we designed a mitochondria-penetrating peptide modified doxorubicin (MPP-Dox) loaded N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer conjugates (PM), as well as a nuclear accumulating HPMA copolymer Dox conjugates (PN) by the nuclear tendency of Dox. After co-delivering the two copolymers (abbreviation for PMN), PM promoted cell apoptosis and inhibited tumor metastasis by damaging mitochondria, whereas PN suppressed cell proliferation and promoted apoptosis by destroying nucleus. Importantly, PM and PN complemented each other as expected. The mitochondrial dysfunction and tumor metastasis inhibition of PM was improved by PN, while cell proliferation suppression and apoptosis by nucleus destroying of PN was enhanced by PM. As a result, tumor growth of breast cancer 4T1 cells in vivo was significantly restrained and lung metastasis was potently decreased and almost eradicated, fully reflecting the advantages of organelle targeting combination therapy. As a consequence, our work showed that concurrent impairment of nucleus and mitochondria was feasible and beneficial to metastatic cancer treatment.

Improving anti-PD-L1 therapy in triple negative breast cancer by polymer-enhanced immunogenic cell death and CXCR4 blockade.

Triple negative breast cancer (TNBC) with highly metastatic features generally does not respond to anti-programmed cell death 1 ligand 1 (PD-L1) therapy due to multiple immunosuppressive mechanisms to exclude and disable T cells. Here, we develop a polymer-based combinatory approach consisting of both immunogenic cell death (ICD)-inducing and CXCR4-inhibiting function to prime tumor microenvironment and improve anti-PD-L1 therapy in TNBC. Our findings revealed that the combination therapy was able to spur the T cell response in primary tumors by increasing the tumor immunogenicity to recruit T cells, removing the physiological barriers of intratumoral fibrosis and collagen to increase T cell infiltration, and reducing the immunosuppressive cells to revive T cells. Meanwhile, such approach efficiently inhibited the formation of pre-metastatic niche in abscopal lung. Because of the significant promotion of anti-tumor and anti-metastasis immunity, the non-responding TNBC gained robust responsiveness to anti-PD-L1 therapy which resulted in complete eradication of orthotopic tumors, inhibition of pulmonary metastasis, and durable memory effects against tumor recurrence. Our work provided a generalizable approach of simultaneous ICD induction and CXCR4 blockade to apply anti-PD-L1 therapy in TNBC.

Circular RNA circ­CCT3 promotes hepatocellular carcinoma progression by regulating the miR­1287­5p/TEAD1/PTCH1/LOX axis.

Hepatocellular carcinoma (HCC) is characterized by a poor prognosis because of its insensitivity to radiation and chemotherapy. Recently, circular RNAs (circRNAs) have been found to serve important roles in hepatocellular carcinogenesis. circ­CCT3, a novel circRNA, was screened from the differential tissue expression results of a circRNA microarray. Relative expression levels of circ­CCT3 in specimens and cell lines were evaluated by reverse transcription­quantitative PCR and the relationship between circ­CCT3 and prognosis was analyzed by Kaplan­Meier curves. The oncogenic role of circ­CCT3 was confirmed in HCC cells through a cell counting kit­8 (CCK­8) assay, a colony formation assay, acridine orange/ethidium bromide double fluorescence staining, flow cytometry, a wound­healing assay and a Transwell assay. Bioinformatics prediction and luciferase reporter assays validated that circ­CCT3 facilitated HCC progression through the miR­1287­5p/TEA domain transcription factor 1 (TEAD1) axis. TEAD1 could then directly activate patched 1 and lysyl oxidase transcription, as analyzed by chromatin immunoprecipitation and luciferase reporter assays. The present study identified a novel circRNA, circ­CCT3, which may be used as a potential therapeutic target for HCC.

Targeting the Opening of Mitochondrial Permeability Transition Pores Potentiates Nanoparticle Drug Delivery and Mitigates Cancer Metastasis.

Mitochondria are highly involved in the metastasis of cancer cells. However, low permeability of mitochondria impedes the entry of anti-cancer drugs. Here, a self-assembled nanoparticle platform is designed that not only targets the DNA-intercalating agent doxorubicin to mitochondria but also enhances the specific penetration by opening the mitochondrial permeability transition pores (MPTPs). With drastic improvement in mitochondrial uptake, the drug delivery system results in substantial mitochondrial impairment leading to amplified induction of apoptosis, depletion of energy supply, and inhibition of numerous metastasis-associated proteins. As a consequence, the drug delivery system significantly inhibits the orthotopic tumor growth, and suppressed the metastasis of cancer cells detached from primary tumors. Additionally, the nanoparticle exhibits a potent effect on eradicating the metastasis of disseminated tumor cell from blood to lung. The results show that strategies of targeting mitochondria and unlocking MPTP are feasible and beneficial to mitigate both tumorigenesis and metastasis.

Combination of mitochondria targeting doxorubicin with Bcl-2 function-converting peptide NuBCP-9 for synergistic breast cancer metastasis inhibition.

Distant organ metastasis is the main cause of death in breast cancer patients. Evidences have shown that mitochondria also play a crucial role in tumor metastasis, except for as apoptosis center. However, the treatment of tumor growth and metastasis was reported to be limited by mitochondria-associated protein Bcl-2, which are gatekeepers of apoptosis and are found to reside in mitochondria mainly. Herein, we designed a mitochondria-targeting doxorubicin delivery system as well as a mitochondrial distributed Bcl-2 function-converting peptide NuBCP-9 delivery system, which are both based on N-(2-hydroxypropyl)methacrylamide copolymers, to achieve a synergistic effect on tumor regression and metastasis inhibition by combination therapy. After mitochondria were damaged by mitochondria-targeting peptide-modified doxorubicin, apoptosis was effectively enhanced by mitochondrial specifically distributed NuBCP-9 peptides, which converted Bcl-2 function from anti-apoptotic to pro-apoptotic and paved the way for the development of mitochondrial impairment. The combination treatment exhibited significant damage to mitochondria, including excess reactive oxygen species (ROS), the permeabilization of mitochondrial outer membrane (MOMP), and apoptosis initiation on 4T1 breast cancer cells. Meanwhile, besides enhanced tumor growth suppression, the combination treatment also improved the inhibition of 4T1 breast cancer metastasis both in vitro and in vivo. By increasing the expression of cytochrome C and decreasing the expression of Bcl-2, metal matrix protease-9 (MMP-9) as well as vascular endothelial growth factor (VEGF), the combination treatment successfully decreased 84% lung metastasis. Overall, our work provided a promising strategy for metastatic cancer treatment through mitochondria-targeting anti-cancer drug delivery and combination with mitochondrial distributed Bcl-2 function-converting peptide.

A novel mitochondrial targeted hybrid peptide modified HPMA copolymers for breast cancer metastasis suppression.

Primary tumor metastasis remains to be a tough obstacle for clinical breast cancer treatment. Since evidences have shown that mitochondria play a crucial role in tumor metastasis, we designed a mitochondrial targeted drug delivery system (P-D-R8MTS) based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers to simultaneously inhibit breast cancer progression and metastasis. A novel mitochondrial targeted hybrid peptide R8MTS, which consists of a cell penetrating peptide octaarginine (R8) and a mitochondrial targeting sequence ALD5MTS, was used as targeting ligand and attached to doxorubicin (DOX) as model drug (DOX-R8MTS). After entering into the tumor cells, DOX-R8MTS was pH-responsibly released from HPMA copolymer backbone in acidic lysosome and efficiently targeted to mitochondria, resulting in enhanced reactive oxygen species (ROS) generation and apoptosis initiation. By destroying mitochondria, P-D-R8MTS not only inhibited cell proliferation but also suppressed migration and invasion of breast cancer 4T1 and MDA-MB-231 cells in vitro. Moreover, P-D-R8MTS exhibited superior inhibition of tumor growth and showed no apparent lung metastatic nodules on 4T1-bearing mice in vivo, which was partially via down-regulation of typical proteins associated with tumor metastasis and invasion: matrix metalloproteinases-2 (MMP-2), vascular endothelial growth factor (VEGF) and transforming growth factor-ß (TGF-ß). Collectively, our work provided a prospectively potential strategy for metastatic cancer treatment through mitochondrial targeted drug delivery.

Enhanced Reactive Oxygen Species Generation by Mitochondria Targeting of Anticancer Drug To Overcome Tumor Multidrug Resistance.

As a major clinical tumor chemotherapeutic burden, multidrug resistance (MDR) is often a result of up-regulation of P-glycoprotein (P-gp), which strongly enhances anticancer drug efflux. The excess mitochondrial reactive oxygen species (ROS) could not only inhibit the function of P-gp through insufficient adenosine triphosphate supply but also cause apoptosis in MDR cells. Here, we designed a mitochondria targeting nanoparticulate system (GNPs-P-Dox-GA) for overcoming MDR through enhanced ROS generation, where increased cellular uptake as well as mitochondria accumulation were both realized by glycyrrhetinic acid (GA). First, doxorubicin was conjugated with GA (GA-Dox) and then grafted onto a N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer backbone via hydrazone bond (P-Dox-GA). The obtained P-Dox-GA was subsequently attached to the surface of gelatin nanoparticles (GNPs). As gelatin is a substrate of tumor extracellular metal matrix protease-2 (MMP2), GNPs-P-Dox-GA nanoparticles could be degraded and release small size P-Dox-GA to facilitate tumor tissue penetration. After P-Dox-GA internalized by tumor cells under GA mediation, Dox-GA detached from HPMA copolymer through hydrolysis of hydrazone bond and then efficiently delivered to mitochondria. Compared to non-GA modified carriers, GNPs-P-Dox-GA exhibited increased cellular uptake nearly 4-fold and mitochondria distribution 8.8-fold, and increased ROS production level nearly 3-fold, significantly decreased efflux rate (55% compared with Dox group) in drug resistant HepG2/ADR cells, and then led to improved in vitro antitumor efficiency in HepG2/ADR cells (IC50 only 19.5% of unmodified ones) as well as exciting in vivo antitumor efficiency on HepG2/ADR heterotopic tumor nude mice (1.75-fold higher tumor growth inhibition rate than free drug).

HPMA Polymeric Nanocarriers for Anticancer Drugs with Tumor Microenvironment-Responsive Extracellular Biodegradation and Intracellular Drug Release.

The balance between systemic toxicity and circulation time for a polymeric nanocarrier to deliver antitumor drugs has been trialed. A new approach to break the balance was proposed in this study by significantly improving its biosafety and prolonging the circulation time, hence, to enhance its anti-tumor efficacy. A matrix metalloproteinases (MMPs)-sensitive peptide (PVGLIGK) was introduced to cross-link the N-(2-hydroxypropyl) methylacrylamide polymer-doxorubicin conjugates (HPMA-Dox) conjugate to construct a nano-size polymeric nanocarrier-Dox assembly (PMD) with a molecular weight (MW) of 73 kDa and this modification has resulted in a prolonged circulation time (a half-time of 20.1 h) and enhanced accumulation of PMD at the tumor site, while negligible systemic toxicity and excellent biocompatibility were displayed after injection of PMD into the mice. The cross-linked nanoassembly was unpacking in the presence of MMPs in the extracellular microenvironment, and the conjugated Dox was released from the nanoassembly in the lysosome/endosome due to an intracellular low pH microenvironment. The released Dox from PMD inhibited tumor cells very efficiently with a tumor growth inhibition of around 70%. The outstanding performance of the dual stimuli-responsive biodegradable polymeric nanocarriers may open a door for other hydrophobic anti-tumor drugs.

Overcoming chemotherapy resistance via simultaneous drug-efflux circumvention and mitochondrial targeting.

Multidrug resistance (MDR) has been considered as a huge challenge to the effective chemotherapy. Therefore, it is necessary to develop new strategies to effectively overcome MDR. Here, based on the previous research of N-(2-hydroxypropyl)methacrylamide (HPMA) polymer-drug conjugates, we designed an effective system that combined drug-efflux circumvention and mitochondria targeting of anticancer drug doxorubicin (Dox). Briefly, Dox was modified with mitochondrial membrane penetrating peptide (MPP) and then attached to (HPMA) copolymers (P-M-Dox). Our study showed that macromolecular HPMA copolymers successfully bypassed drug efflux pumps and escorted Dox into resistant MCF-7/ADR cells via endocytic pathway. Subsequently, the mitochondria accumulation of drugs was significantly enhanced with 11.6-fold increase by MPP modification. The excellent mitochondria targeting then resulted in significant enhancement of reactive oxygen species (ROS) as well as reduction of adenosine triphosphate (ATP) production, which could further inhibit drug efflux and resistant cancer cell growth. By reversing Dox resistance, P-M-Dox achieved much better suppression in the growth of 3D MCF-7/ADR tumor spheroids compared with free Dox. Hence, our study provides a promising approach to treat drug-resistant cancer through simultaneous drug efflux circumvention and direct mitochondria delivery.

Long non-coding RNA LOXL1-AS1 acts as a ceRNA for miR-324-3p to contribute to cholangiocarcinoma progression via modulation of ATP-binding cassette transporter A1.

Accumulating evidence has appreciated long non-coding RNAs (lncRNAs) as novel prognostic markers and therapeutic targets in malignant carcinomas. Here, we aim to investigate the value of a novel cancer-related lncRNA, LOXL1-AS1, in cholangiocarcinoma (CCA). LOXL1-AS1 was found overexpressed in CCA tissues screened by high-throughput sequencing technology. Upregulation of LOXL1-AS1 was identified by TCGA database and qRT-PCR analysis. Additionally, upregulation of LOXL1-AS1 was associated with lymph node invasion, advanced TNM stages and unfavorable prognosis. Loss-of-function and gain-of-function experiments were conducted and validated that LOXL1-AS1 could facilitate cell proliferation, migration and invasion and attenuate cell apoptosis. Moreover, luciferase reporter and rescue assays indicated that LOXL1-AS1 functioned as a ceRNA to elevate ATP-binding cassette transporter A1 (ABCA1) level by sponging miR-324-3p and exhibited the malignant phenotypes of CCA cells, thereby playing an oncogenic role in CCA. Taken together, this study reveals that LOXL1-AS1 might act as a potential biomarker and therapeutic target for CCA clinical application.

Multifunctional Nanoparticles Enable Efficient Oral Delivery of Biomacromolecules via Improving Payload Stability and Regulating the Transcytosis Pathway.

In oral delivery of biomacromolecules, ligand-modified nanoparticles (NPs) have emerged as a promising tool to improve the epithelial uptake of the loaded protein/peptide. Unfortunately, the stability and the transport mechanisms of the biotherapeutics during the intracellular transportation still remained unclear, leading to the poor transepithelial efficiency. Additionally, developing novel approaches to simultaneously monitor the payload bioactivity during the transport processes is veritably benefit for keeping their bioactivity. In the present study, EGP peptide (KRKKKGKGLGKKRDPCLRKYK), a ligand with high affinity to heparan sulfate proteoglycans (HSPGs), was found remarkably increasing the cellular uptake (4.5-fold) and also surprisingly achieving high transcytosis efficiency (4.2-fold) of poly(lactide- co-glycolide) NPs on Caco-2 cell monolayer. Compared with unmodified NPs (C NPs), EGP modified NPs (EGP NPs) exhibited more desirable colloidal stability within epithelia. In the subsequent study, the bioactivity of encapsulated insulin during the cellular transportation was innovatively monitored by a glucose consumption assay. Inspiringly, EGP NPs could mostly retain the bioactivity of loaded insulin whereas insulin from INS-C NPs was significantly degraded. Then the detailed mechanism study revealed that the binding of EGP to HSPGs played a vital role on NP transportation. Unlike C NPs being delivered in the endo/lysosomal pathway, EGP NPs were involved in caveolae-mediated transport, which contributes to the efficient avoidance of the lysosomal entrapment and sequentially facilitates the direct apical-to-basolateral transcytosis. The enhanced absorption of EGP NPs was confirmed in in situ intestinal loop models. Most importantly, oral administrated INS-EGP NPs generated a strong hypoglycemic response on diabetic rats with 10.2-fold and 2.6-fold increase in bioavailability compared with free insulin and INS-C NPs, respectively. The work provided an innovative strategy to monitor the payload bioactivity during the transport processes and proposed a novel aspect to increase oral bioavailability of biomacromolecules via improving payload stability and regulating the transcytosis pathway of nanocarriers.

Synergistic enhancement of anticancer therapeutic efficacy of HPMA copolymer doxorubicin conjugates via combination of ligand modification and stimuli-response srategies.

N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymer has been extensively studied as drug carrier for tumor therapy. Due to the Enhanced Permeability and Retention (EPR) effect, HPMA copolymer drug conjugates are able to be passively accumulated in the tumor site. Currently, efficient uptake of this polymeric system by the cancer cells remains a big challenge, as HPMA polymer is highly hydrophilic, neutrally charged, and has low affinity towards cell membrane. In this study, selective and enhanced intracellular internalization of the copolymer-drug conjugates was achieved by utilizing a hybrid strategy including ligand modification and stimuli response. This hybrid approach was rationally designed to comprise cationic HPMA copolymer backbone as drug carrier, doxorubicin (Dox) as model drug, hydrazone bond as drug spacer, FQSIYPpIK (FQS) peptide as αvß3 targeting ligand and 2, 3-Dimethylmaleic Anhydride (DMA) as a shielded/deshielded cationic group. We demonstrated our system exhibited the "seek-and-destroy" tumor tropic behavior by sequentially undergoing the following steps: (i) tumor passive targeting mediated by EPR effect; (ii) charge reversal at tumor extracellular pH of 6.5; (iii) synergistically enhanced cell uptake via electrostatic interaction with cell membrane and FQS ligand-mediated bio-recognition; (iv) drug released in the lysosome; and v) anticancer effect exerted by the targeted delivery of the Dox.