Cargo-selective and adaptive delivery of nucleic acid therapeutics by bola-amphiphilic dendrimers.

Nucleic acid therapeutics are becoming an important drug modality, offering the unique opportunity to address "undruggable" targets, respond rapidly to evolving pathogens, and treat diseases at the gene level for precision medicine. However, nucleic acid therapeutics have poor bioavailability and are chemolabile and enzymolabile, imposing the need for delivery vectors. Dendrimers, by virtue of their well-defined structure and cooperative multivalence, represent precision delivery systems. We synthesized and studied bola-amphiphilic dendrimers for cargo-selective and on-demand delivery of DNA and small interfering RNA (siRNA), both important nucleic acid therapeutics. Remarkably, superior performances were achieved for siRNA delivery with the second-generation dendrimer, yet for DNA delivery with the third generation. We systematically studied these dendrimers with regard to cargo binding, cellular uptake, endosomal release, and in vivo delivery. Differences in size both of the dendrimers and their nucleic acid cargos impacted the cooperative multivalent interactions for cargo binding and release, leading to cargo-adaptive and selective delivery. Moreover, both dendrimers harnessed the advantages of lipid and polymer vectors, while offering nanotechnology-based tumor targeting and redox-responsive cargo release. Notably, they allowed tumor- and cancer cell-specific delivery of siRNA and DNA therapeutics for effective treatment in different cancer models, including aggressive and metastatic malignancies, outperforming the currently available vectors. This study provides avenues to engineer tailor-made vectors for nucleic acid delivery and precision medicine.

Targeted degradation of oncogenic BCR-ABL by silencing the gene of NEDD8 E3 ligase RAPSYN.

Tyrosine kinase inhibitors have been the standard treatment for patients with Philadelphia chromosome-positive (Ph+) leukemia. However, a series of issues, including drug resistance, relapse and intolerance, are still an unmet medical need. Here, we report the targeted siRNA-based lipid nanoparticles in Ph+ leukemic cell lines for gene therapy of Ph+ leukemia, which specifically targets a recently identified NEDD8 E3 ligase RAPSYN in Ph+ leukemic cells to disrupt the neddylation of oncogenic BCR-ABL. To achieve the specificity for Ph+ leukemia therapy, a single-chain fragment variable region (scFv) of anti-CD79B monoclonal antibody was covalently conjugated on the surface of OA2-siRAPSYN lipid nanoparticles to generate the targeted lipid nanoparticles (scFv-OA2-siRAPSYN). Through effectively silencing RAPSYN gene in leukemic cell lines by the nanoparticles, BCR-ABL was remarkably degraded accompanied by the inhibition of proliferation and the promotion of apoptosis. The specific targeting, therapeutic effects and systemic safety were further evaluated and demonstrated in cell line-derived mouse models. The present study has not only addressed the clinical need of Ph+ leukemia, but also enabled gene therapy against a less druggable target.

Recognition, Intervention, and Monitoring of Neutrophils in Acute Ischemic Stroke.

Neutrophils are implicated in numerous inflammatory diseases, and especially in acute ischemic stroke (AIS). The unchecked migration of neutrophils into cerebral ischemic regions, and their subsequent release of reactive oxygen species, are considered the primary causes of reperfusion injury following AIS. Reducing the infiltration of inflammatory neutrophils may therefore be a useful therapy for AIS. Here, inspired by the specific cell-cell recognition that occurs between platelets and inflammatory neutrophils, we describe platelet-mimetic nanoparticles (PTNPs) that can be used to directly recognize, intervene, and monitor inflammatory neutrophils in the AIS treatment and therapeutic evaluation. We demonstrate that PTNPs, coloaded with piceatannol, a selective spleen tyrosine kinase inhibitor, and superparamagnetic iron oxide (SPIO), a T2 contrast agent, can successfully recognize adherent neutrophils via platelet membrane coating. The loaded piceatannol could then be delivered to adherent neutrophils and detach them into circulation, thus decreasing neutrophil infiltration and reducing infarct size. Moreover, when coupled with magnetic resonance imaging, internalized SPIO could be used to monitor the inflammatory neutrophils, associated with therapeutic effects, in real time. This approach is an innovative method for both the treatment and therapeutic evaluation of AIS, and provides new insights into how to treat and monitor neutrophil-associated diseases.

Neoadjuvant Chemotherapy Based on Abraxane/Human Neutrophils Cytopharmaceuticals with Radiotherapy for Gastric Cancer.

Gastric cancer remains one of the most lethal cancers with high incidence and mortality worldwide. The majority of gastric cancer patients are those who have first been diagnosed in advanced stage, in which the standard chemo-radiotherapy produces limited benefit along with severe general toxicity, thus the demand for improved therapeutic efficacy and decreased side effects drives the development of novel therapeutic strategies. Here, a neoadjuvant chemotherapy based on Abraxane/human neutrophils (NEs) cytopharmaceuticals with radiotherapy is presented for effective cancer treatment. Human NEs, the most abundant white blood cells in peripheral blood, are developed to carry Abraxane, the commercial albumin-bound paclitaxel nanoparticle, to form cytopharmaceuticals (Abraxane/NEs) which have been confirmed to maintain the intrinsic functions of human NEs. The modest radiation is applied not only to exert tumor disruption, but also to increase the release of inflammatory factors which guide the NEs homing to the tumoral sites. These amplified inflammatory factors at tumor sites excessively activate Abraxane/NEs to form neutrophil extracellular traps, along with a burst release of Abraxane to induce superior tumor suppression. This adjuvant chemo-radiotherapy based on cytopharmaceuticals may provide new opportunities for advanced cancer treatment, which reveals the huge clinical potential of human neutrophils as drug delivery vectors.

Design, synthesis and evaluation of pyrrolo[2,3-d]pyrimidine-phenylamide hybrids as potent Janus kinase 2 inhibitors.

Janus kinase 2 (JAK2) plays an essential role in the signaling of hormone-like cytokines and growth factors, which has been convinced as an important target of myeloproliferative neoplasms (MPNs) therapy. In this study, a series of novel pyrrolo[2,3-d]pyrimidine-phenylamide hybrids were designed and synthesized as potential JAK2 inhibitors through hybridization strategy. In vitro biological studies showed that most of these compounds exhibited potent activity against JAK2. Especially, compound 16c was identified as a suitable lead compound, which showed favorable pharmacokinetic profiles in rats (F=73.57%), excellent in vitro efficacy against erythroleukemic cells (TF-1, IC50=0.14µM), and high selectivity for JAK2 (IC50=6nM with >97-fold selectivity vs JAK3).

Sequential intra-intercellular nanoparticle delivery system for deep tumor penetration.

To achieve deep tumor penetration of large-sized nanoparticles (NPs), we have developed a reversible swelling-shrinking nanogel in response to pH variation for a sequential intra-intercellular NP delivery. The nanogel had a crosslinked polyelectrolyte core, consisting of N-lysinal-N'-succinyl chitosan and poly(N-isopropylacrylamide), and a crosslinked bovine serum albumin shell, which was able to swell in an acidic environment and shrink back under neutral conditions. The swelling resulted in a rapid release of the encapsulated chemotherapeutics in the cancer cells for efficient cytotoxicity. After being liberated from the dead cells, the contractive nanogel could infect neighboring cancer cells closer to the center of the tumor tissue.

Reprogrammed siTNFα/neutrophil cytopharmaceuticals targeting inflamed joints for rheumatoid arthritis therapy.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by severe synovial inflammation and cartilage damage. Despite great progress in RA therapy, there still lacks the drugs to completely cure RA patients. Herein, we propose a reprogrammed neutrophil cytopharmaceuticals loading with TNFα-targeting-siRNA (siTNFα) as an alternative anti-inflammatory approach for RA treatment. The loaded siTNFα act as not only the gene therapeutics to inhibit TNFα production by macrophages in inflamed synovium, but also the editors to reprogram neutrophils to anti-inflammatory phenotypes. Leveraging the active tendency of neutrophils to inflammation, the reprogrammed siTNFα/neutrophil cytopharmaceuticals (siTNFα/TP/NEs) can rapidly migrate to the inflamed synovium, transfer the loaded siTNFα to macrophages followed by the significant reduction of TNFα expression, and circumvent the pro-inflammatory activity of neutrophils, thus leading to the alleviated synovial inflammation and improved cartilage protection. Our work provides a promising cytopharmaceutical for RA treatment, and puts forward a living neutrophil-based gene delivery platform.

New-generation cytopharmaceuticals with powerfully boosted extravasation for enhanced cancer therapy.

Effective extravasation of therapeutic agents into solid tumors still faces huge challenges. Since the doubted effectiveness of enhanced penetration and retention effect, first-generation neutrophil cytopharmaceuticals with encapsulated drugs have been developed to improve the drug accumulation in tumors based on the active chemotaxis and extravasation of neutrophils. Herein, a new generation of neutrophil cytopharmaceuticals with enhanced tumor-specific extravasation is reported to satisfy more complex clinical demands. This neutrophil cytopharmaceutical is obtained by anchoring vascular endothelial growth factor receptor 2 (VEGFR2)-targeting peptide K237 on neutrophil membrane after endocytosis of chemotherapeutics by neutrophils. Leveraging the cytokine-mediated active migration of neutrophils, the specific-recognition of K237 peptide to tumor vascular endothelium expedites the migration and enhances tight adhesion of neutrophils to vascular endothelium, thus improving the extravasation of therapeutic agents to target sites. Moreover, anti-angiogenesis effect from VEGFR2-blocking by K237 peptide achieves a cooperative tumor destruction with cytotoxic effects from released chemotherapeutics. This study demonstrates the great potential of enhanced proactive extravasation of cytopharmaceuticals via a cell-anchoring technology, leading to expedited drug infiltration and boosted therapeutic effects, which can be applied in other cell therapies to improve efficacy.

Sensitizing Tumors to Immune Checkpoint Blockage via STING Agonists Delivered by Tumor-Penetrating Neutrophil Cytopharmaceuticals.

Immune checkpoint inhibitors (ICIs) have displayed potential efficacy in triple-negative breast cancer (TNBC) treatment, while only a minority of patients benefit from ICI therapy currently. Although activation of the innate immune stimulator of interferon genes (STING) pathway potentiates antitumor immunity and thus sensitizes tumors to ICIs, the efficient tumor penetration of STING agonists remains critically challenging. Herein, we prepare a tumor-penetrating neotype neutrophil cytopharmaceutical (NEs@STING-Mal-NP) with liposomal STING agonists conjugating on the surface of neutrophils, which is different from the typical neutrophil cytopharmaceutical that loads drugs inside the neutrophils. We show NEs@STING-Mal-NP that inherit the merits of neutrophils including proactive tumor vascular extravasation and tissue penetration significantly boost the tumor penetration of STING agonists. Moreover, the backpacked liposomal STING agonists can be released in response to hyaluronidase rich in the tumor environment, leading to enhanced uptake by tumor-infiltrating immune cells and tumor cells. Thus, NEs@STING-Mal-NP effectively activate the STING pathway and reinvigorate the tumor environment through converting macrophages and neutrophils to antitumor phenotypes, promoting the maturation of dendritic cells, and enhancing the infiltration and tumoricidal ability of T cells. Specifically, this cytopharmaceutical displays a significant inhibition on tumor growth and prolongs the survival of TNBC-bearing mice when combined with ICIs. We demonstrate that neutrophils serve as promising vehicles for delivering STING agonists throughout solid tumors and the developed neutrophil cytopharmaceuticals with backpacked STING agonists exhibit huge potential in boosting the immunotherapy of ICIs.

An EPR-Independent extravasation Strategy: Deformable leukocytes as vehicles for improved solid tumor therapy.

Effective delivery of therapeutic modality throughout the tumorous nidus plays a crucial role in successful solid tumor treatment. However, conventional nanomedicines based on enhanced permeability and retention (EPR) effect have yielded limited delivery/therapeutic efficiency, due mainly to the heterogeneity of the solid tumor. Leukocytes, which could intrinsically migrate across the vessel wall and crawl through tissue interstitium in a self-deformable manner, have currently emerged as an alternative drug delivery vehicle. In this review, we start with the intrinsic properties of leukocytes (e.g., extravasation and crawling inside tumor), focusing on unveiling the conceptual rationality of leveraging leukocytes as EPR-independent delivery vehicles. Then we discussed various cargoes-loading/unloading strategies for leukocyte-based vehicles as well as their promising applications. This review aims to serve as an up-to-date compilation, which might provide inspiration for scientists in the field of drug delivery.

Single-component lipid nanoparticles for engineering SOCS1 gene-silenced dendritic cells to boost tumor immunotherapy.

Dendritic cells (DCs) that can prime antitumor responses show great potential in tumor immunotherapy, whereas the unsatisfactory effect which can be ascribed in part to the high expression of inhibitory cytokines, such as the suppressor of cytokine signaling 1 (SOCS1), restricts their application. Thus, silencing these genes in DCs is essential for DC-based therapy. However, safe and effective delivery of siRNA to DCs still faces challenges. Herein, we designed single-component lipid nanoparticles comprising a solely cationic lipid (OA2) for introducing siRNA into mouse DCs in order to inhibit the immunosuppressive gene and boost the effector responses of DC-based therapy. Compared to other multi-component lipid nanoparticles, single-component lipid nanoparticles are theoretically easy-to-control and detective, which is beneficial for future translation. We showed that the application of OA2 lipid nanoparticles significantly downregulated the expression of SOCS1 in DCs over 50%, compared with the commercial lipofectine2000. Besides, the treatment of OA2 lipid nanoparticles had no influence on the antigen capture of DCs. Thus, we fabricated a SOCS1-downregulated DC vaccine pulsed with Ova antigen and demonstrated that the antigen presentation and pro-inflammatory factor secretion ability of DCs were improved due to the SOCS1 downregulation, leading to an ameliorated immunosuppressive tumor microenvironment and finally exhibiting potent tumor prevention and suppression in B16-Ova tumor-bearing mice. Single-component lipid nanoparticles, which provide an available vector platform for siRNA delivery to primary DCs, appear to be a potent tool to engineer DCs and in turn boost DC-based tumor immunotherapy.

Neutrophil Cyto-Pharmaceuticals Suppressing Tumor Metastasis via Inhibiting Hypoxia-Inducible Factor-1α in Circulating Breast Cancer Cells.

Circulating tumor cells (CTCs) are reported as the precursor of tumor metastases, implying that stifling CTCs would be beneficial for metastasis prevention. However, challenges remain for the application of therapies that aim at CTCs due to lack of effective CTC-targeting strategy and sensitive therapeutic agents. Herein, a general CTC-intervention strategy based on neutrophil cyto-pharmaceuticals is proposed for suppressing CTC colonization and metastasis formation. Breast cancer 4T1 cells are infused as the mimic CTCs, and 4T1 cells trapped are first elucidated in neutrophil extracellular traps (NETs) expressing high levels of hypoxia-inducible factor-1α (HIF-1α) due to NET formation and thus promoting tumor cell colonization through enhanced migration, invasion and stemness. After verifying HIF-1α as a potential target for metastasis prevention, living neutrophil cyto-pharmaceuticals (CytPNEs) loaded with HIF-1α inhibitor are fabricated to therapeutically inhibit HIF-1α. It is demonstrated that CytPNEs can specially convey the HIF-1α inhibitor to 4T1 cells according to the inflammatory chemotaxis of neutrophils and down-regulate HIF-1α, thereby inhibiting metastasis and prolonging the median survival of mice bearing breast cancer lung metastasis. The research offers a new perspective for understanding the mechanism of CTC colonization, and puts forward the strategy of targeted intervention of CTCs as a meaningful treatment for tumor metastasis.

A neutrophil-mimetic magnetic nanoprobe for molecular magnetic resonance imaging of stroke-induced neuroinflammation.

Neuroinflammation plays a key role in the progression of brain injury induced by stroke, and has become a promising target for therapeutic intervention for stroke. Monitoring this pivotal process of neuroinflammation is highly desirable to guide specific therapy. However, there is still a lack of a satisfactory nanoprobe to selectively monitor neuroinflammation. As endothelial cell activation is a hallmark of neuroinflammation, it would be clinically relevant to develop a non-invasive in vivo imaging technique to detect the endothelial activation process. Herein, inspired by the specific neutrophil-endothelium interaction, we designed neutrophil-camouflaged magnetic nanoprobes (NMNPs) that can be used to target activated endothelial cells for improved neuroinflammation imaging. NMNPs are composed of an inner core of superparamagnetic iron oxide (SPIO)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles and a biomimetic outer shell of a neutrophil membrane, which maintained the biocompatibility and targeting ability of neutrophils and the excellent contrast effects of SPIO. Moreover, we demonstrated that NMNPs can successfully bind to inflamed cerebral vasculature using the intravital imaging of live cerebral microvessels in transient middle cerebral artery occlusion (tMCAO) mice. After that, NMNPs could further accumulate in the brain vasculature and exhibit excellent contrast effects for stroke-induced neuroinflammation and biosafety. We believe that the neutrophil-camouflaged magnetic nanoprobe could serve as a highly safe and selective nanoprobe for neuroinflammation imaging and has alluring prospects for clinical application.

Modular synthesis of amphiphilic chitosan derivatives based on copper-free click reaction for drug delivery.

Amphiphilic chitosan derivatives have attracted wide attention as drug carriers due to their physicochemical properties. However, obtaining a desired amphiphilic chitosan derivative by tuning the various functional groups was complex and time-consuming. Therefore, a facile and common synthesis strategy would be promising. In this study, a modular strategy based on strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction was designed and applied in synthesizing deoxycholic acid- or octanoic acid-modified N-azido propionyl-N,O-sulfate chitosan through tuning the hydrophobic groups. Additionally, chitosan derivatives with the same substitute groups were prepared via amide coupling as controls. We demonstrated that these derivates via the two strategies showed no obvious difference in physicochemical properties, drug loading ability and biosafety, indicating the feasibility of modular strategy. Notably, the modular strategy exhibited advantages including high reactivity, flexibility and reproducibility. We believe that this modular strategy could provide varied chitosan derivatives in an easy and high-efficiency way for improving multifunctional drug carriers.

Cytopharmaceuticals: An emerging paradigm for drug delivery.

Cytopharmaceuticals, in which drugs/nanomedicines are loaded into/onto autologous patient- or allogeneic donor-derived living cells ex vivo, have displayed great promise for targeted drug delivery in terms of improved biocompatibility, superior targeting, and prolonged circulation. Despite certain impressive therapeutic benefits in preclinical studies, several obstacles retard their clinical application, such as the lack of facile and convenient methods of carrier cell acquisition, technologies for preparing cytopharmaceuticals at scale with undisturbed carrier cell viability, and modalities for monitoring the in vivo fate of cytopharmaceuticals. To comprehensively understand cytopharmaceuticals and thereby accelerate their clinical translation, this review covers the main sources of various cytopharmaceuticals, technologies for preparing cytopharmaceuticals, the in vivo fate of cytopharmaceuticals including carrier cells and loaded drugs/nanomedicines, and the application prospects of cytopharmaceuticals. It is our hope that this review will elucidate the bottlenecks associated with cytopharmaceutical preparation, leading to the acceleration of future industrialization of cell-based formulations.

Anisamide-functionalized pH-responsive amphiphilic chitosan-based paclitaxel micelles for sigma-1 receptor targeted prostate cancer treatment.

Controlled release and tumor-selective distribution are highly desirable for anticancer nanomedicines. Here, we design and synthesize an anisamide-conjugated N-octyl-N,O-maleoyl-O-phosphoryl chitosan (a-OMPC) which can form amphiphilic micelles featuring pH-responsive release and high affinity to sigma-1 receptor-overexpressed tumors for paclitaxel (PTX) delivery. Thereinto, maleoyl and phosphoryl groups cooperatively contribute to pH-responsive drug release due to a conversion from hydrophile to hydrophobe in the acidic microenvironment of endo/lysosomes. We demonstrated that PTX-loaded a-OMPC micelles (PTX-aM) enhanced the cellular internalization via the affinity between anisamide and sigma-1 receptor, rapidly released drug in endo/lysosomes and elevated the cytotoxicity against PC-3 cells. The in vivo studies further verified that PTX-aM could largely accumulate at the tumor site even after 24 h of administration, resulting in obvious inhibition effect and prolonged survival period in PC-3 tumor xenograft-bearing mice. Moreover, OMPC showed no obvious hemolytic and acute toxicity. Collectively, this chitosan derivate holds a promising potential in application of prostate cancer-targeted drug delivery system.

Combination of metabolic intervention and T cell therapy enhances solid tumor immunotherapy.

Treatment of solid tumors with T cell therapy has yielded limited therapeutic benefits to date. Although T cell therapy in combination with proinflammatory cytokines or immune checkpoints inhibitors has demonstrated preclinical and clinical successes in a subset of solid tumors, unsatisfactory results and severe toxicities necessitate the development of effective and safe combinatorial strategies. Here, the liposomal avasimibe (a metabolism-modulating drug) was clicked onto the T cell surface by lipid insertion without disturbing the physiological functions of the T cell. Avasimibe could be restrained on the T cell surface during circulation and extravasation and locally released to increase the concentration of cholesterol in the T cell membrane, which induced rapid T cell receptor clustering and sustained T cell activation. Treatment with surface anchor-engineered T cells, including mouse T cell receptor transgenic CD8+ T cells or human chimeric antigen receptor T cells, resulted in superior antitumor efficacy in mouse models of melanoma and glioblastoma. Glioblastoma was completely eradicated in three of the five mice receiving surface anchor-engineered chimeric antigen receptor T cells, whereas mice in other treatment groups survived no more than 64 days. Moreover, the administration of engineered T cells showed no obvious systemic side effects. These cell-surface anchor-engineered T cells hold translational potential because of their simple generation and their safety profile.

The development of tertiary amine cationic lipids for safe and efficient siRNA delivery.

Cationic liposomes have shown great potential in efficient siRNA delivery, and their positive charge is crucial for tight extracellular siRNA binding, effective intracellular siRNA disassembly and physiological toxicity. Thus, the development of novel cationic lipids with a suitable positive charge is desirable for safe and efficient siRNA delivery. Herein, we fabricated a library of 21 tertiary amine-derived cationic lipids (TA) to achieve a balance between effectiveness and safe siRNA delivery. The screened TA13 liposomes, which consisted of TA13 and helper lipid DOPE at a mole ratio of 1 : 1, readily condensed siRNA to form lipoplexes (TA13 LPs), achieving stronger gene silencing in diverse cells than the commercially available vector Lipo2000. Moreover, the TA13 LPs demonstrated effective in vivo gene silencing and good safety in normal mice. The improved gene silencing efficiency of the TA13 LPs is ascribed to their capability of sequentially conquering the barriers met by in vivo siRNA delivery. Notably, the TA13 LPs delivered ApoB-siRNA and obviously decreased ApoB mRNA expression in the liver and the total cholesterol and low-density lipoprotein in the serum of hypercholesterolemia mice, indicating a potential siRNA therapeutic for hypercholesterolemia treatment. It is anticipated that these novel tertiary amine-based liposomes can provide a simple and widely-used platform for the safe and effective delivery of siRNA, and their structure-activity relationships can aid in the further development of effective cationic lipids.

Solubility and bioavailability enhancement study of lopinavir solid dispersion matrixed with a polymeric surfactant - Soluplus.

As a biopharmaceutical classification system Class IV drug, lopinavir (LPV) shows relatively poor water solubility and permeation in vivo. In the study, we developed novel solid dispersions (SD) of LPV to improve its bioavailability and to describe their overall behaviors. By employing solvent evaporation for a preliminary formulation screening, the SDs of LPV-polymer-sorbitan monolaurate (SBM, as the wetting agent) at 1:4:0.4 (w/w) dramatically enhanced the LPV dissolution in a non-sink medium, and then hot-melt extrusion (HME) was applied to improve the dissolution further. A hydrophilic polymer - Kollidon VA 64 (VA64) and a polymeric surfactant Soluplus were employed as matrix respectively in the optimized formulations. The dissolution profiles of extrudates were significantly higher than those of SDs prepared with solvent-evaporation method. It was attributed to the stronger intermolecular interactions between LPV and the polymers in the HME process, which was also supported by the stability analysis after 6 months storage under 25 °C/60% RH. The differential scanning calorimetry, fourier transform infrared spectroscopy and equilibrium studies showed VA64 only created hydrogen bonding (H-bond) with LPV, but Soluplus generated both H-bond and micelle thanks to its amphiphilic structure. In addition, the bioavailability of LPV in Soluplus matrixed extrudate was 1.70-fold of VA64 matrixed extrudate and 3.70-fold of LPV crystal. In situ permeability and Caco-2 cell transport studies revealed that Soluplus significantly enhanced the permeability of LPV through rat intestine and Caco-2 cell monolayers by P-glycoprotein (P-gp) inhibition. Herein, Soluplus matrixed extrudate improved the LPV bioavailability through three mechanisms: H-bond with LPV, micelle formation in water and P-gp inhibition in vivo. These unique advantages of Soluplus suggested it is a promising carrier for poorly water soluble drugs, especially the substrates of P-gp.