Motility and tumor infiltration are key aspects of invariant natural killer T cell anti-tumor function.

Dysfunction of invariant natural killer T (iNKT) cells contributes to immune resistance of tumors. Most mechanistic studies focus on their static functional status before or after activation, not considering motility as an important characteristic for antigen scanning and thus anti-tumor capability. Here we show via intravital imaging, that impaired motility of iNKT cells and their exclusion from tumors both contribute to the diminished anti-tumor iNKT cell response. Mechanistically, CD1d, expressed on macrophages, interferes with tumor infiltration of iNKT cells and iNKT-DC interactions but does not influence their intratumoral motility. VCAM1, expressed by cancer cells, restricts iNKT cell motility and inhibits their antigen scanning and activation by DCs via reducing CDC42 expression. Blocking VCAM1-CD49d signaling improves motility and activation of intratumoral iNKT cells, and consequently augments their anti-tumor function. Interference with macrophage-iNKT cell interactions further enhances the anti-tumor capability of iNKT cells. Thus, our findings provide a direction to enhance the efficacy of iNKT cell-based immunotherapy via motility regulation.

Breaking through the basement membrane barrier to improve nanotherapeutic delivery to tumours.

An effective nanotherapeutic transport from the vasculature to the tumour is crucial for cancer treatment with minimal side effects. Here we demonstrate that, in addition to the endothelial barrier, the tumour vascular basement membrane surrounding the endothelium acts as a formidable mechanical barrier that entraps nanoparticles (NPs) in the subendothelial void, forming perivascular NP pools. Breaking through this basement membrane barrier substantially increases NP extravasation. Using inflammation triggered by local hyperthermia, we develop a cooperative immunodriven strategy to overcome the basement membrane barrier that leads to robust tumour killing. Hyperthermia-triggered accumulation and inflammation of platelets attract neutrophils to the NP pools. The subsequent movement of neutrophils through the basement membrane can release the NPs entrapped in the subendothelial void, resulting in increased NP penetration into deeper tumours. We show the necessity of considering the tumour vascular basement membrane barrier when delivering nanotherapeutics. Understanding this barrier will contribute to developing more effective antitumour therapies.

Nanomedicines with high drug availability and drug sensitivity overcome hypoxia-associated drug resistance.

Tumor hypoxia heterogeneity, a hallmark of the tumor microenvironment, confers resistance to conventional chemotherapy due to insufficient drug availability and drug sensitivity in hypoxic regions. To overcome these challenges, we develope a nanomedicine, NPHPaPN, constructed with hyaluronic acid (HA) grafted with cisplatin prodrug and PEG-azobenzene for hypoxia-responsive PEG shell deshielding and loaded with a DNA damage repair inhibitor (NERi). After arriving at the tumor site, NPHPaPN deshields the PEG shell in response to hypoxia due to the enzymolysis of azobenzene and thus exposes HA. The exposed HA binds to the highly expressed CD44 on cisplatin-resistant tumor cells and mediates drug internalization, thus increasing drug availability to hypoxic tumor cells. After intracellular hyaluronidase-mediated cleavage, the HA NPs release the cisplatin prodrug and NERi, and cause enhanced DNA damage and consequent cell death, thus enhancing the drug sensitivity of hypoxic tumor cells. Eventually, NPHPaPN achieves distinct tumor growth suppression with an ∼84.4% inhibition rate.

Enhanced Intracellular Transcytosis of Nanoparticles by Degrading Extracellular Matrix for Deep Tissue Radiotherapy of Pancreatic Adenocarcinoma.

Intracellular transcytosis can enhance the penetration of nanomedicines to deep avascular tumor tissues, but strategies that can improve transcytosis are limited. In this study, we discovered that pyknomorphic extracellular matrix (ECM) is a shield that impairs endocytosis of nanoparticles and their movement between adjacent cells and thus limits their active transcytosis in tumors. We further showed that degradation of pivotal constituent of ECM (i.e., collagen) effectively enhances intracellular transcytosis of nanoparticles. Specifically, a collagenase conjugating transcytosis nanoparticle (Col-TNP) can dissociate into collagenase and cationized gold nanoparticles in response to tumor acidity, which enables their ECM tampering ability and active transcytosis in tumors. The breakage of ECM further enhances the active transcytosis of cationized nanoparticles into deep tumor tissues as well as radiosensitization efficacy of pancreatic adenocarcinoma. Our study opens up new paths to enhance the active transcytosis of nanomedicines for the treatment of cancers and other diseases.

Pre- and post-irradiation mild hyperthermia enabled by NIR-II for sensitizing radiotherapy.

The clinical application of cancer radiotherapy is critically impeded by hypoxia-induced radioresistance, insufficient DNA damage, and multiple DNA repair mechanisms. Herein we demonstrate a dual-hyperthermia strategy to potentiate radiotherapy by relieving tumor hypoxia and preventing irradiation-induced DNA damage repair. The tumor hyperthermia temperature was well-controlled by a near infrared laser with minimal side effects using PEGylated nanobipyramids (PNBys) as the photo-transducer. PNBys have narrow longitudinal localized surface plasmon resonance peak in NIR-II window with a high extinction coefficient (2.0 × 1011 M-1 cm-1) and an excellent photothermal conversion efficiency (44.2%). PNBys-induced mild hyperthermia (MHt) prior to radiotherapy enables vessel dilation, blood perfusion, and hypoxia relief, resulting in an increased susceptibility of tumor cells response to radiotherapy. On the other hand, MHt after radiotherapy inhibits the repair of DNA damage generated by irradiation. The PNBys exert hierarchically superior antitumor effects by the combination of MHt pre- and post-radiotherapy in murine mammary tumor EMT-6 model. Consequently, different from the simple combination of RT and MHt, the coupling of pre- and post-MHt with RT by PNBys open intriguing avenues towards new promising antitumor efficacy.

Self-Reporting and Splitting Nanopomegranates Potentiate Deep Tissue Cancer Radiotherapy via Elevated Diffusion and Transcytosis.

The efficacy of nanoradiosensitizers in cancer therapy has been primarily impeded by their limited accessibility to radioresistant cancer cells residing deep inside tumor tissues. The failure to report tumor response to radiotherapy generally delays adjustment of the treatment schedule and sets up another substantial obstacle to clinical success. Here, we develop a nanopomegranate (RNP) platform that not only visualizes the cancer radiosensitivities but also potentiates deep tissue cancer radiotherapy via elevated passive diffusion and active transcytosis. The RNPs are engineered through the programmed self-assembly of a tumor environment-targeting polymeric matrix and modular building blocks of ultrasmall gold nanoparticles (Au5). Once RNPs reach the tumors, the environmental acidity triggers the splitting and surface cationization of Au5. The small dimension of Au5 allows its passive diffusion, while positive surface charge enables its active transcytosis to cross the tumor interstitium. Meanwhile, the reporter element monitors the feedback of favorable radiotherapy responsiveness by detecting the activated apoptosis after radiation. The pivotal role of RNPs in improving and identifying radiotherapeutic outcomes is demonstrated in various tumor bearing mouse models with different radiosensitivities. In summary, our strategy offers a promising paradigm for deep tissue drug delivery as well as individualized precision radiotherapy.

Polyphosphoestered Nanomedicines with Tunable Surface Hydrophilicity for Cancer Drug Delivery.

The surface hydrophilicity of nanoparticles has a major impact on their biological fates. Ascertaining the correlation between nanoparticle surface hydrophilicity and their biological behaviors is particularly instructive for future nanomedicine design and their antitumor efficacy optimization. Herein, we designed a series of polymeric nanoparticles based on polyphosphoesters with well-controlled surface hydrophilicity in the molecular level and systemically evaluated their biological behaviors. The results demonstrated that high surface hydrophilicity preferred lower protein absorption, better stability, longer blood circulation, and higher tumor accumulation but lower cellular uptake. Upon encapsulation of drugs, nanoparticles with high hydrophilicity showed an excellent antitumor therapeutic efficacy in both primary and metastatic tumors as compared to the relatively hydrophobic ones. Further analyses revealed that the superior antitumor outcome was attributed to the balance of tumor accumulation and cellular uptake, demonstrating the particular importance of nanoparticle surface hydrophilicity regulation on the antitumor efficacy. Our work provides a potent guideline for a rational designation on the surface hydrophilicity of nanoparticles for cancer treatment optimization.

Near-Infrared II Phototherapy Induces Deep Tissue Immunogenic Cell Death and Potentiates Cancer Immunotherapy.

The deep and inner beds of solid tumors lack lymphocytic infiltration and are subjected to various immune escape mechanisms. Reversing immunosuppression deep within the tumor is vital in clinical cancer therapy, however it remains a huge challenge. In this work, we have demonstrated the use of a second window near-infrared (NIR(II)) photothermal treatment to trigger more homogeneous and deeper immunogenic cancer cell death in solid tumors, thereby eliciting both innate and adaptive immune responses for tumor control and metastasis prevention. Specifically, photothermal transducers with similar components, structures, and photothermal conversion efficiencies, but different absorptions in red light, NIR(I), and NIR(II) biowindows, were constructed by controlling the self-assembly of gold nanoparticles on fluidic liposomes. In vitro, photothermal treatments induced immunogenic cell death (ICD) that were accompanied by the release of damage-associated molecular patterns (DAMPs) regardless of the wavelength of incident lasers. In vivo, NIR(II) light resulted in a more homogeneous release and distribution of DAMPs in the deeper parts of the tumors. With the induction of ICD, NIR(II) photothermal therapy simultaneously triggered both innate and adaptive immune responses and enabled efficient tumor control with 5/8 of the mice remaining tumor-free in the cancer vaccination assay. Additionally, the NIR(II) photothermal treatment in combination with checkpoint blockade therapy exerted long-term tumor control over both primary and distant tumors. Finally, using systemically administered two-dimensional polypyrrole nanosheets as a NIR(II) transducer, we achieved striking therapeutic effects against whole-body tumor metastasis via a synergistic photothermal-immunological response.

Enzyme-Activatable Interferon-Poly(α-amino acid) Conjugates for Tumor Microenvironment Potentiation.

Protein-polymer conjugation is a clinically validated approach to enhanced pharmacokinetic properties. However, the permanent attachment of polymers often leads to irreversibly reduced protein bioactivity and poor tissue penetration. As such, the use of protein-polymer conjugates for solid tumors remains elusive. Herein, we report a simple strategy using enzyme-activatable and size-shrinkable protein-polypeptide conjugates to overcome this clinical challenge. Briefly, a matrix metalloproteinase (MMP)-responsive peptide sequence is introduced between a therapeutic protein interferon (IFN) and a synthetic polypeptide P(EG3Glu)20. The resulting site-specific MMP-responsive conjugate, denoted as PEP20-M-IFN, can, therefore, release the attached P(EG3Glu)20 to achieve both protein activation and deep penetration into the tumor microenvironment (TME). Compared to a similarly produced nonresponsive analogue conjugate PEP20-IFN, our results find PEP20-M-IFN to show higher bioactivity in vitro, improved tumor retention, and deeper penetration in a MMP2-dependent manner. Moreover, systemic administration of PEP20-M-IFN shows outstanding antitumor efficacy in both OVCAR3 and SKOV3 ovarian tumor models in mice. This work highlights the releasable PEPylation strategy for protein drug potentiation at the TME and opens up new opportunities in clinics for the treatment of malignant solid tumors.

Nanoclustered Cascaded Enzymes for Targeted Tumor Starvation and Deoxygenation-Activated Chemotherapy without Systemic Toxicity.

Intratumoral glucose depletion-induced cancer starvation represents an important strategy for anticancer therapy, but it is often limited by systemic toxicity, nonspecificity, and adaptive development of parallel energy supplies. Herein, we introduce a concept of cascaded catalytic nanomedicine by combining targeted tumor starvation and deoxygenation-activated chemotherapy for an efficient cancer treatment with reduced systemic toxicity. Briefly, nanoclustered cascaded enzymes were synthesized by covalently cross-linking glucose oxidase (GOx) and catalase (CAT) via a pH-responsive polymer. The release of the enzymes can be first triggered by the mildly acidic tumor microenvironment and then be self-accelerated by the subsequent generation of gluconic acid. Once released, GOx can rapidly deplete glucose and molecular oxygen in tumor cells while the toxic side product, i.e., H2O2, can be readily decomposed by CAT for site-specific and low-toxicity tumor starvation. Furthermore, the enzymatic cascades also created a local hypoxia with the oxygen consumption and reductase-activated prodrugs for an additional chemotherapy. The current report represents a promising combinatorial approach using cascaded catalytic nanomedicine to reach concurrent selectivity and efficiency of cancer therapeutics.

Tumor Reoxygenation and Blood Perfusion Enhanced Photodynamic Therapy using Ultrathin Graphdiyne Oxide Nanosheets.

Both diffusion-limited and perfusion-limited hypoxia are associated with tumor progression, metastasis, and the resistance to therapeutic modalities. A strategy that can efficiently overcome both types of hypoxia to enhance the efficacy of cancer treatment has not been reported yet. Here, it is shown that by using biomimetic ultrathin graphdiyne oxide (GDYO) nanosheets, both types of hypoxia can be simultaneously addressed toward an ideal photodynamic therapy (PDT). The GDYO nanosheets, which are oxidized and exfoliated from graphdiyne (GDY), are able to efficiently catalyze water oxidation to release O2 and generate singlet oxygen (1O2) using near-infrared irradiation. Meanwhile, GDYO nanosheets also exhibit excellent light-to-heat conversion performance with a photothermal conversion efficiency of 60.8%. Thus, after the GDYO nanosheets are coated with iRGD peptide-modified red blood membrane (i-RBM) to achieve tumor targeting, the biomimetic GDYO@i-RBM nanosheets can simultaneously enhance tumor reoxygenation and blood perfusion for PDT. This study provides new insights into utilizing novel water-splitting materials to relieve both diffusion- and perfusion-limited hypoxia for the development of a novel therapeutic platform.

Cancer Chemoradiotherapy Duo: Nano-Enabled Targeting of DNA Lesion Formation and DNA Damage Response.

Both production of DNA damage and subsequent prevention of its repair are crucial in concluding the therapeutic outcome of radiotherapy (RT). However, nearly all current strategies for improving RT focus only on one of the two aspects and overlook the necessity of their combinations. In this work, we introduce a concept of DNA-dual-targeting nanomedicine (NM) to simultaneously enhance DNA lesion formation and prevent the succeeding repair. Briefly, the cisplatin prodrug loaded in NM can form platinated DNA in cell nuclei, making DNA more vulnerable to the ionizing radiation generated by RT. Concomitantly, the spatial-temporally codelivered vorinostat, a histone deacetylase inhibitor, prolongs the build-up of double-strand breaks and causes cell apoptosis en masse, probably due to the suppressed expression of DNA repair proteins. Furthermore, this nanoplatform is suitable for fluorescence and magnetic resonance imaging techniques, enabling accurate trafficking of the NM as well as reliable real-time imaging-guided precision RT. Finally, results from in vitro and in vivo jointly reveal that this dual-action system attains a remarkably enhanced radiotherapeutic outcome. In conclusion, our imaging-guided DNA-dual-targeting design represents a novel strategy for efficient cancer precision RT.

Hierarchical Multiplexing Nanodroplets for Imaging-Guided Cancer Radiotherapy via DNA Damage Enhancement and Concomitant DNA Repair Prevention.

Clinical success of cancer radiotherapy is usually impeded by a combination of two factors, i.e., insufficient DNA damage and rapid DNA repair during and after treatment, respectively. Existing strategies for optimizing the radiotherapeutic efficacy often focus on only one facet of the issue, which may fail to function in the long term trials. Herein, we report a DNA-dual-targeting approach for enhanced cancer radiotherapy using a hierarchical multiplexing nanodroplet, which can simultaneously promote DNA lesion formation and prevent subsequent DNA damage repair. Specifically, the ultrasmall gold nanoparticles encapsulated in the liquid nanodroplets can concentrate the radiation energy and induce dramatic DNA damage as evidenced by the enhanced formation of γ-H2AX foci as well as in vivo tumor growth inhibition. Additionally, the ultrasound-triggered burst release of oxygen may relieve tumor hypoxia and fix the DNA radical intermediates produced by ionizing radiation, prevent DNA repair, and eventually result in cancer death. Finally, the nanodroplet platform is compatible with fluorescence, ultrasound, and magnetic resonance imaging techniques, allowing for real-time in vivo imaging-guided precision radiotherapy in an EMT-6 tumor model with significantly enhanced treatment efficacy. Our DNA-dual-targeting design of simultaneously enhancing DNA damage and preventing DNA repair presents an innovative strategy to effective cancer radiotherapy.

Oxygen self-sufficient fluorinated polypeptide nanoparticles for NIR imaging-guided enhanced photodynamic therapy.

A major hindrance for photodynamic therapy (PDT) to achieve higher efficiency is the hypoxia environment in the tumor area and the PDT-induced continuous consumption of molecular oxygen. Oxygen self-sufficient fluorinated polypeptide nanoparticles have been synthesized via the loading of a NIR photosensitizer (BODIPY-Br2) into a water-dispersible drug delivery system for high efficiency PDT. As a result of the higher oxygen capacity and 1O2 lifetime enhancement of perfluorocarbon, the whole PDT agent demonstrated higher oxygen uptake and enhanced singlet oxygen production, showing the potential to improve the PDT efficacy in hypoxia tumor environments after light irradiation. In vitro studies including cellular uptake and PDT efficiency were evaluated using hepatocellular carcinoma HepG2 cells as models, and the enhanced PDT efficiency of fluorinated polypeptide DDS with higher O2 content was measured on a tumor-bearing BALB/c mice model by in vivo experiments. Results demonstrated that the fluorinated polypeptide platform plays a significant role as an effective delivery vehicle for small molecule photosensitizers while increasing the generation of reactive oxygen species (ROS) and having higher cytotoxicity to cancer cells, especially in the hypoxia environment. In addition, the BODIPY-Br2 photosensitizer worked for both PDT and imaging in the NIR region making it a potential theranostic for cancer treatment.