Stepwise Ultra-pH-Sensitive Micelles Overcome a pKa Barrier for Systemic Lymph Node Delivery.

While local nanoparticle delivery to lymph nodes is well studied, there are few design criteria for intravenous delivery to the entire lymph node repertoire. In this study, we investigated the effect of NP pH transition on lymph node targeting by employing a series of ultra-pH-sensitive (UPS) polymeric micelles. The UPS library responds to pH thresholds (pKa 6.9, 6.2, and 5.3) over a range of physiological pH. We observed a dependence of intravenous lymph node targeting on micelle pH transition. UPS6.9 (subscript indicates pKa) shows poor lymph node delivery, while UPS5.3 delivers efficiently to lymph node sets. We investigated targeting mechanisms of UPS5.3, observing an accumulation among lymph node lymphatics and a dependence on lymph node-resident macrophages. To overcome the pH-threshold barrier, which limits UPS6.9, we rationally designed a nanoparticle coassembly of UPS6.9 with UPS5.3, called HyUPS. The HyUPS micelle retains the constitutive pH transitions of each polymer, showing stepwise responses to discrete pH thresholds. We demonstrate that HyUPS improves UPS6.9 delivery to lymph nodes, extending this platform for disease detection of lymph node metastasis.

Isoliquiritigenin Containing PH Sensitive Micelles for Enhanced Anti-Colitis Activity.

Isoliquiritigenin (ISL) is known to have a variety of pharmacological activities, but its poor water solubility limits its application. In order to improve the bioavailability of ISL and its anti-colitis activity, this study aims to develop an effective drug delivery system loaded with ISL. In this study, ISL pH-sensitive micelles (ISL-M) were prepared by thin film hydration method. The micellar size (PS), polydispersity index (PDI), electrokinetic potential (ζ-potential), drug loading (DL), encapsulation rate (EE) and other physical parameters were characterized. The storage stability of ISL-M was tested, release in vitro and pharmacokinetic studies in rats were performed, and the anti-inflammatory effect of ISL-M on ulcerative colitis induced by dextran sulfate sodium (DSS) was evaluated. The results showed that PS, PDI, ZP, EE% and DL% of ISL-M were 151.15±1.04 nm, 0.092±0.014, -31.32±0.721 mV, 93.97±1.53 % and 8.42±0.34 %, respectively. Compared with unformulated ISL (F-ISL), the cumulative release rate of ISL-M in the three different media was significantly increased and showed a certain pH sensitivity. The area under drug curve (AUC0-t) and peak concentration (Cmax) of ISL-M group were 2.94 and 4.06 times higher than those of ISL group. In addition, ISL-M is expected to develop new methods for increasing the bioavailability and anti-inflammatory activity of ISL.

Exploring the impact of severity in hepatic fibrosis disease on the intrahepatic distribution of novel biodegradable nanoparticles targeted towards different disease biomarkers.

Nanoparticle-based drug delivery systems (DDS) have shown promising results in reversing hepatic fibrosis, a common pathological basis of chronic liver diseases (CLDs), in preclinical animal models. However, none of these nanoparticle formulations has transitioned to clinical usage and there are currently no FDA-approved drugs available for liver fibrosis. This highlights the need for a better understanding of the challenges faced by nanoparticles in this complex disease setting. Here, we have systematically studied the impact of targeting strategy, the degree of macrophage infiltration during fibrosis, and the severity of fibrosis, on the liver uptake and intrahepatic distribution of nanocarriers. When tested in mice with advanced liver fibrosis, we demonstrated that the targeting ligand density plays a significant role in determining the uptake and retention of the nanoparticles in the fibrotic liver whilst the type of targeting ligand modulates the trafficking of these nanoparticles into the cell population of interest - activated hepatic stellate cells (aHSCs). Engineering the targeting strategy indeed reduced the uptake of nanoparticles in typical mononuclear phagocyte (MPS) cell populations, but not the infiltrated macrophages. Meanwhile, additional functionalization may be required to enhance the efficacy of DDS in end-stage fibrosis/cirrhosis compared to early stages.

Elucidation of Protonation Cooperativity of a STING-Activating Polymer.

Stimuli-responsive nanomaterials have the potential to improve the performance and overcome existing barriers of conventional nanotherapeutics. Molecular cooperativity design in stimuli-responsive nanomedicine can amplify physiological signals, enabling a cooperative response for improved diagnostic and therapeutic precision. Previously, this work reported an ultra-pH-sensitive polymer, PEG-b-PC7A, that possesses innate immune activating properties by binding to the stimulator of interferon genes (STING) through polyvalent phase condensation. This interaction enhances STING activation and synergizes with the endogenous STING ligand for robust cancer immunotherapy. Despite its successes in innate immune activation, the fundamental physicochemical and pH-responsive properties of PC7A require further investigation. Here, this study elucidates the protonation cooperativity driven by the phase transition of PC7A copolymer. The highly cooperative system displays an "all-or-nothing" proton distribution between highly charged unimer (all) and neutral micelle (nothing) states without gradually protonated intermediates. The binary protonation behavior is further illustrated in pH-precision-controlled release of a representative anticancer drug, ß-lapachone, by PC7A micelles over a noncooperative PE5A polymer. Furthermore, the bimodal distribution of protons is represented by a high Hill coefficient (nH  > 9), featuring strong positive cooperativity. This study highlights the nanoscale pH cooperativity of an immune activating polymer, providing insights into the physicochemical characterization and design parameters for future nanotherapeutics development.

Endosomal Escape of Bioactives Deployed via Nanocarriers: Insights Into the Design of Polymeric Micelles.

Cytoplasmic delivery of bioactives requires the use of strategies such as active transport, electroporation, or the use of nanocarriers such as polymeric nanoparticles, liposomes, micelles, and dendrimers. It is essential to deliver bioactive molecules in the cytoplasm to achieve targeted effects by enabling organelle targeting. One of the biggest bottlenecks in the successful cytoplasmic delivery of bioactives through nanocarriers is their sequestration in the endosomes that leads to the degradation of drugs by progressing to lysosomes. In this review, we discussed mechanisms by which nanocarriers are endocytosed, the mechanisms of endosomal escape, and more importantly, the strategies that can be and have been employed for their escape from the endosomes are summarized. Like other nanocarriers, polymeric micelles can be designed for endosomal escape, however, a careful control is needed in their design to balance between the possible toxicity and endosomal escape efficiency. Keeping this in view, polyion complex micelles, and polymers that have the ability to escape the endosome, are fully discussed. Finally, we provided some perspectives for designing the polymeric micelles for efficient cytoplasmic delivery of bioactive agents through endosomal escape.

TfR-T12 short peptide and pH sensitive cell transmembrane peptide modified nano-composite micelles for glioma treatment via remodeling tumor microenvironment.

Two kinds of amphiphilic block copolymers of TfR-T12-PEG-PLGA and TATH7-PEG-PLGA were synthesized to self-assembly nano-composite micelles for encapsulating paclitaxel and imiquimod synchronously. TfR-T12 peptide modified nano-composite micelles can pass through BBB in a TfR-mediated way to achieve targeted delivery of chemotherapeutic drugs, and pH sensitive TATH7 peptide modified nano-composite micelles enhanced uptake efficiency more significantly under pH 5.5 medium than pH 7.4 medium. The results of pharmacodynamic evaluation in vivo showed that the nano-composite micelles had achieved good anti-tumor effect in subcutaneous and normotopia glioma models, and effectively prolonged the life cycle of tumor-bearing mice. The nano-composite micelles regulated the immunosuppression phenomenon of tumor microenvironment significantly, and promoted the M1 polarization of TAMs, then enhanced the proliferation and activation of CD8+ T cells in tumor microenvironment. It comes to conclusion that the nano-composite micelle achieves the purpose of effective treatment of glioma by chemotherapy combined with immunotherapy.

Stimuli-responsive, dual-function prodrug encapsulated in hyaluronic acid micelles to overcome doxorubicin resistance.

Multidrug resistance (MDR) is the main challenge faced by cancer chemotherapy. Drug-conjugate offers a promising strategy for breast cancer therapy. In this regard, we developed a DNVM multifunctional drug delivery system by crosslinking doxorubicin (DOX) and vitamin E succinate (VES) with a pH-sensitive hydrazone bond and then encapsulated the DOX-NN-VES prodrug into pH-sensitive hyaluronic acid-2-(octadecyloxy)-1,3-dioxan-5-amine (HOD) micelles. DOX resistant MCF-7/ADR cell were adopted as a model to study the capability and mechanism of MDR reversal. DNVM exhibited much higher cytotoxicity and cell uptake efficiency compared with that of acid-insensitive DOX-VES loaded HOD micelles (DVSM) and DOX loaded HOD micelles (DOXM), indicating the better capacity of DNVM for the reversal of MDR. Moreover, DNVM prevented drug efflux more effectively, inhibited the expression of P-gp, induced excessive production of reactive oxygen species and affected the expression of apoptosis-related proteins. In vivo experiments showed that DNVM significantly inhibited the tumor growth with no obvious changes in the body weight of MCF-7/ADR cells-bearing nude mice. The results suggested that the "double gain" DNVM can synergistically enhance the efficacy of chemotherapeutics for DOX resistant tumor cells and has the potential to overcome tumor MDR. STATEMENT OF SIGNIFICANCE: A dual-functional pH-sensitive doxorubicin - vitamin E succinate prodrug was developed and loaded into tumor microenvironment-sensitive hyaluronic acid-2-(octadecyloxy)-1,3-dioxan-5-amine micelle system (DNVM) for sequencing stimuli-release and overcoming doxorubicin resistance. The "double gain" DNVM can synergistically enhance the efficacy of chemotherapeutics for doxorubicin resistant tumor cells and has the potential to overcome tumor multiple drug resistance.

Tumor-Targeted Inhibition of Monocarboxylate Transporter 1 Improves T-Cell Immunotherapy of Solid Tumors.

Export of lactic acid from glycolytic cancer cells to the extracellular tumor milieu has been reported to enhance tumor growth and suppress antitumor immunity. In this study, a pH-activatable nanodrug is reported for tumor-targeted inhibition of monocarboxylate transporter-1 (MCT1) that reverses lactic acid-induced tumor immunosuppression. The nanodrug is composed of an MCT1 inhibitor (AZD3965) loaded inside the ultra-pH-sensitive nanoparticles (AZD-UPS NPs). AZD-UPS NP is produced by a microfluidics method with improved drug loading efficiency and optimal nanoparticle size over sonication methods. The nanodrug remains as intact micelles at pH 7.4 but rapidly disassembles and releases payload upon exposure to acidic pH. When combined with anti-PD-1 therapy, AZD-UPS NP leads to potent tumor growth inhibition and increases survival in two tumor models over oral administration of AZD3965 at dramatically reduced dose (>200-fold). Safety evaluations demonstrate reduced drug distribution in heart and liver tissues with decrease in toxic biomarkers such as cardiac troponin by the nanodrug. Increased T-cell infiltration and reduced exhaustive PD1+ Tim3+ T cells are found in tumors. These data illustrate that tumor-targeted inhibition of MCT1 can reverse the immune suppressive microenvironment of solid tumors for increased safety and antitumor efficacy of cancer immunotherapy.

Antigen folding improves loading efficiency and antitumor efficacy of PC7A nanoparticle vaccine.

Cancer vaccines hold great promise to produce antigen-specific T cell immunity for personalized therapy of cancer. Previously, we reported an ultra-pH-sensitive nanoparticle, PC7A, capable of priming an efficacious immune response without significant systemic toxicity. Despite the early success, the relationship between antigen properties and encapsulation efficiency for downstream immune activation remains poorly understood. In this study, we investigated a small library of melanoma antigens and the effects of several formulation methods on the efficiency of peptide loading inside PC7A nanoparticles. Results show loading efficiency is not highly dependent on the formulation methods, but instead mainly driven by the peptide antigen properties. In particular, we identified a phase transition event, namely the folding of antigenic peptides from random coils to α-helical structure, is important for antigen loading inside PC7A nanoparticles. Mutation of a peptide that abrogates the formation of helical structure resulted in poor loading efficiency. Antitumor efficacy studies in melanoma-bearing mice demonstrate the importance of peptide loading in vaccine-induced antitumor immunity. This study highlights the contribution of phase transition of peptide antigens on vaccine formulation in order to make widespread use of personalized nanoparticle vaccines feasible.

Exploiting nanoscale cooperativity for precision medicine.

Precise spatiotemporal control of molecular transport is vital to functional physiological systems. Nature evolved to apply macromolecular cooperativity to achieve precision over systemic delivery of important molecules. In drug delivery, conventional nanocarriers employ inert materials and rely on passive accumulation for tissue targeting and diffusion for drug release. Early clinical studies show these nanodrugs have not delivered the anticipated impact on therapy. Inspired by nature, we propose a design principle that incorporates nanoscale cooperativity and phase transition to sense and amplify physiological signals to improve the therapeutic outcome. Using ultra-pH-sensitive (UPS) nanoparticles as an example, we demonstrate how all-or-nothing protonation cooperativity during micelle assembly/disassembly can be exploited to increase dose accumulation and achieve rapid drug release in acidic microenvironments. In a separate study, we show the effectiveness of a single polymer composition to accomplish cytosolic delivery of tumor antigens with activation of stimulator of interferon genes (STING) in lymph node-resident dendritic cells for cancer immunotherapy. Molecular cooperativity is a hallmark of nanobiology that offers a valuable strategy to functionalize nanomedicine systems to achieve precision medicine.

Drug Delivery System Based on pH-Sensitive Biocompatible Poly(2-vinyl pyridine)-b-poly(ethylene oxide) Nanomicelles Loaded with Curcumin and 5-Fluorouracil.

Smart polymeric micelles (PMs) are of practical interest as nanocarriers for the encapsulation and controlled release of hydrophobic drugs. Two hydrophobic drugs, naturally-based curcumin (Cur) and synthetic 5-fluorouracil (5-FU), were loaded into the PMs formed by a well-defined pH-sensitive poly(2-vinyl pyridine)-b-poly(ethylene oxide) (P2VP90-b-PEO398) block copolymer. The influence of the drug loading on the micellar sizes was investigated by dynamic light scattering (DLS) and it appears that the size of the PMs increases from around 60 to 100 nm when Cur is loaded. On the contrary, the loading of the 5-FU has a smaller effect on the micellar sizes. This difference can be attributed to higher molar mass of Cur with respect to 5-FU but also to higher loading efficiency of Cur, 6.4%, compared to that of 5-FU, 5.8%. In vitro drug release was studied at pH 2, 6.8, and 7.4, and it was observed that the pH controls the release of both drugs. At pH 2, where the P2VP sequences from the "frozen-in" micellar core are protonated, the drug release efficiencies exceed 90%. Moreover, it was demonstrated, by in vitro assays, that these PMs are hemocompatible and biocompatible. Furthermore, the PMs protect the Cur against the photo-degradation, whereas the non-ionic PEO corona limits the adsorption of bovine serum albumin (BSA) protein on the surface. This study demonstrates that these pH-sensitive PMs are suitable for practical utilization as human-safe and smart, injectable drug delivery systems.

Degradable double hydrophilic block copolymers and tripartite polyionic complex micelles thereof for small interfering ribonucleic acids (siRNA) delivery.

Polymer vectors for gene therapy have been largely investigated as an alternative to viral vectors. In particular, double hydrophilic block copolymers (DHBCs) have shown potential in this domain, but to date studies mainly focus on non-degradable copolymers, which may be a restriction for further development. To overcome this limitation, we synthesized a DHBC (PEG43-b-PCL12(COOH)6.5) composed of a poly(ethylene glycol) (PEG) non-ionic and bioeliminable block and a degradable carboxylic acid-functionalized poly(ε-caprolactone) (PCL) block. The potential of this DHBC as an original vector for small interfering ribonucleic acids (siRNA) to formulate tripartite polyionic complex (PIC) micelles with poly(lysine) (PLL) was evaluated. We first studied the impact of the charge ratio (R) on the size and the zeta potential of the resulting micelles. With a charge ratio R = 1, one formulation with optimized physico-chemical properties showed the ability to complex 75% of siRNA. We showed a stability of the micelles at pH 7.4 and a disruption at pH 5, which allowed a pH-triggered siRNA release and proved the pH-stimuli responsive character of the tripartite micelles. In addition, the tripartite PIC micelles were shown to be non-cytotoxic below 40 µg/mL. The potential of these siRNA vectors was further evaluated in vitro: it was found that the tripartite PIC micelles allowed siRNA internalization to be 3 times higher than PLL polyplexes in murine mesenchymal stem cells, and were able to transfect human breast cancer cells. Overall, this set of data pre-validates the use of degradable DHBC as non-viral vectors for the encapsulation and the controlled release of siRNA, which may therefore constitute a sound alternative to non-degradable and/or cytotoxic polycationic vectors.

Calcitriol-Loaded Dual-pH-Sensitive Micelle Counteracts Pro-Metastasis Effect of Paclitaxel in Triple-Negative Breast Cancer Therapy.

The therapy of triple-negative breast cancer (TNBC) relies on chemotherapy basing on cytotoxic agents, including paclitaxel (PTX). Unfortunately, PTX will facilitate the invasion of cancer cells and the formation of metastases. To counteract pro-metastasis of PTX in TNBC therapy, in this work, calcitriol (CTL) is delivered along with PTX by a dual-pH-sensitive micelle. The PTX/CTL-co-loaded dual-pH-sensitive micelle (PCDM) can switch its surface charge from negative to positive at the tumor tissue and release PTX and CTL inside the lysosomes because of the structure change of the polymers composing PCDM under the acidic condition. This property makes PCDM able to escape from mononuclear-phagocyte system clearance and easy to enter tumor cells. PCDM efficiently suppresses the 4T1 primary tumor growth in mice and inhibits lung metastasis, due to downregulation of matrix metalloproteinase-9 and BCL-2 levels, upregulation of E-cadherin level, and counteracting the PTX-induced elevation of C-C motif chemokine ligand 2 (CCL2) and Ly6C+ monocytes levels by CTL. PCDM shows good biocompatibility without promoting the serum calcium level. Therefore, the combination of PTX and CTL based on this pH-sensitive micelle is promising for the TNBC treatment.

Harnessing the Formation of Natural Killer-Tumor Cell Immunological Synapses for Enhanced Therapeutic Effect in Solid Tumors.

The formation of an immunological synapse (IS) on recognition of a cancer cell is the main mechanism underlying the natural killer (NK)-cell-mediated killing of tumor cells. Herein, an integrative strategy for cancer therapy against solid tumors is reported, in which alterations in the cleft of IS, following the secretion of acidic granular content, are utilized as a trigger for the delivery of chemotherapeutic drugs. NK cells are decorated with the IS-environment-responsive micellar system to ensure the release of the payload when they attack cancer cells. Using this strategy, the immunological cytotoxic killing effect of NK cells against solid tumors is reinforced with the site-specific diffusion of chemotherapeutic agents. Harnessing the intrinsic mechanism for the recognition of abnormal cells and the tumor-homing effect of NK cells limit the adverse systemic effects of chemotherapeutic drugs. This approach may provide a pragmatic platform for the universal and effective utilization of IS formation.

Targeting the Oncogene KRAS Mutant Pancreatic Cancer by Synergistic Blocking of Lysosomal Acidification and Rapid Drug Release.

Survival of KRAS mutant pancreatic cancer is critically dependent on reprogrammed metabolism including elevated macropinocytosis, autophagy, and lysosomal degradation of proteins. Lysosomal acidification is indispensable to protein catabolism, which makes it an exploitable metabolic target for KRAS mutant pancreatic cancer. Herein we investigated ultra-pH-sensitive micelles (UPSM) with pH-specific buffering of organelle pH and rapid drug release as a promising therapy against pancreatic cancer. UPSM undergo micelle-unimer phase transition at their apparent p Ka, with dramatically increased buffer capacity in a narrow pH range (<0.3 pH). Cell studies including amino acid profiling showed that UPSM inhibited lysosomal catabolism more efficiently than conventional lysosomotropic agents ( e. g., chloroquine) and induced cell apoptosis under starved condition. Moreover, pH-triggered rapid drug release from triptolide prodrug-loaded UPSM (T-UPSM) significantly enhanced cytotoxicity over non-pH-sensitive micelles (T-NPSM). Importantly, T-UPSM demonstrated superior safety and antitumor efficacy over triptolide and T-NPSM in KRAS mutant pancreatic cancer mouse models. Our findings suggest that the ultra-pH-sensitive nanoparticles are a promising therapeutic platform to treat KRAS mutant pancreatic cancer through simultaneous lysosomal pH buffering and rapid drug release.

Mechanisms of drug release in pH-sensitive micelles for tumour targeted drug delivery system: A review.

During the past decades, chemotherapy has been regarded as the most effective method for tumor therapy, but still faces significant challenges, such as poor tumor selectivity and multidrug resistance. The development of targeted drug delivery systems brings certain dramatic advantages for reducing the side effects and improving the therapeutic efficacy. Coupling a specific stimuli-triggered drug release mechanism with these delivery systems is one of the most prevalent approaches for targeted therapy. Among these approaches, pH-sensitive micelles are regarded as the most general strategy with advantages of increasing solubility of water-insoluble drugs, pH-sensitive release, high drug loading, etc. This review will focus on the potential of pH-sensitive micelles in tumor therapy, analyze four types of drug-loaded micelles and mechanisms of drug release and give an exhaustive collection of recent investigations. Sufficient understanding of these mechanisms will help us to design more efficient pH-sensitive drug delivery system to address the challenges encountered in targeted drug delivery systems for tumor therapy.

Preparation and evaluation of poly(l-histidine) based pH-sensitive micelles for intracellular delivery of doxorubicin against MCF-7/ADR cells.

In this study, a pH-sensitive micelle self-assembled from poly(l-histidine) based triblock copolymers of poly(ethylene glycol)-poly(d,l-lactide)-poly(l-histidine) (mPEG-PLA-PHis) was prepared and used as the intracellular doxorubicin (Dox) delivery for cancer chemotherapy. Dox was loaded into the micelles by thin-film hydration method and a Box-Behnken design for three factors at three levels was used to optimize the preparations. The optimized mPEG-PLA-Phis/Dox micelles exhibited good encapsulation efficiency of 91.12%, a mean diameter of 45 nm and narrow size distribution with polydispersity index of 0.256. In vitro drug release studies demonstrated that Dox was released from the micelles in a pH-dependent manner. Furthermore, the cellular evaluation of Dox loaded micelles displayed that the micelles possessed high antitumor activity in vitro with an IC50 of 35.30 µg/ml against MCF-7/ADR cells. The confocal microscopy and flow cytometry experiments indicated that mPEG-PLA-Phis micelles mediated efficient cytoplasmic delivery of Dox with the aid of poly(l-histidine) mediated endosomal escape. In addition, blank mPEG-PLA-Phis micelles were shown to be nontoxic to MCF-7/ADR cells even at a high concentration of 200 µg/ml. The pH-sensitive mPEG-PLA-PHis micelles have been demonstrated to be a promising nanosystem for the intracellular delivery of Dox for MDR reversal.

pH-sensitive polymeric micelles formed by doxorubicin conjugated prodrugs for co-delivery of doxorubicin and paclitaxel.

A doxorubicin conjugated prodrug incorporated acid-sensitive linkage between drug and Pluronic F127-chitosan (F127-CS) polymer was successfully synthesized. Subsequently a pH-sensitive polymeric micelle system was designed based on the conjugated prodrugs (F127-CS-DOX) to co-deliver doxorubicin and paclitaxel. Paclitaxel (PTX) was physically entrapped in the hydrophobic inner core of the micelles simultaneously. The structures of conjugates were analyzed by means of (1)H NMR and UV-vis spectrum. Size distribution and morphology of the micelles were observed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The results indicated that obtained micelles had good dispersity and the diameter was between 56.3 and 403.4 nm. The loading of PTX into the micelle increased with higher DOX content. DOX and PTX release from polymeric micelles followed an acid-triggered manner. Furthermore, in vivo pharmacokinetic study also showed that the area under the plasma concentration time curve (AUC0-∞) values of PTX and DOX for PTX-loaded F127-CS-DOX micelles in rats were 3.97 and 4.38-fold higher than those for PTX plus DOX solution. These results suggested the PTX-loaded F127-CS-DOX micelles would be a promising carrier for co-delivering DOX and PTX.