The prevention and treatment of cardiovascular diseases (CVD) has largely focused on lowering circulating LDL cholesterol, yet a significant burden of atherosclerotic disease remains even when LDL is low. Recently, microRNAs (miRNAs) have emerged as exciting therapeutic targets for cardiovascular disease. miRNAs are small noncoding RNAs that post-transcriptionally regulate gene expression by degradation or translational inhibition of target mRNAs. A number of miRNAs have been found to modulate all stages of atherosclerosis, particularly those that promote the efflux of excess cholesterol from lipid-laden macrophages in the vessel wall to the liver. However, one of the major challenges of miRNA-based therapy is to achieve tissue-specific, efficient, and safe delivery of miRNAs in vivo. We sought to develop chitosan nanoparticles (chNPs) that can deliver functional miRNA mimics to macrophages and to determine if these nanoparticles can alter cholesterol efflux and reverse cholesterol transport in vivo. We developed chNPs with a size range of 150−200 nm via the ionic gelation method using tripolyphosphate (TPP) as a cross-linker. In this method, negatively charged miRNAs were encapsulated in the nanoparticles by ionic interactions with polymeric components. We then optimized the efficiency of intracellular delivery of different formulations of chitosan/TPP/miRNA to mouse macrophages. Using a well-defined miRNA with roles in macrophage cholesterol metabolism, we tested whether chNPs could deliver functional miRNAs to macrophages. We find chNPs can transfer exogenous miR-33 to nai ̈ve macrophages and reduce the expression of ABCA1, a potent miR-33 target gene, both in vitro and in vivo, confirming that miRNAs delivered via nanoparticles can escape the endosomal system and function in the RISC complex. Because miR-33 and ABCA1 play a key role in regulating the efflux of cholesterol from macrophages, we also confirmed that macrophages treated with miR-33-loaded chNPs exhibited reduced cholesterol efflux to apolipoprotein A1, further confirming functional delivery of the miRNA. In vivo, mice treated with miR33-chNPs showed decreased reverse cholesterol transport (RCT) to the plasma, liver, and feces. In contrast, when efflux-promoting miRNAs were delivered via chNPs, ABCA1 expression and cholesterol efflux into the RCT pathway were improved. Over all, miRNAs can be efficiently delivered to macrophages via nanoparticles, where they can function to regulate ABCA1 expression and cholesterol efflux, suggesting that these miRNA nanoparticles can be used in vivo to target atherosclerotic lesions.

Three-dimensional (3D) optical microscopy can be used to understand and improve the delivery of nanomedicine. However, this approach cannot be performed for analyzing liposomes in tissues because the processing step to make tissues transparent for imaging typically removes the lipids. Here, we developed a tag, termed REMNANT, that enables 3D imaging of organic materials in biological tissues. We demonstrated the utility of this tag for the 3D mapping of liposomes in intact tissues. We also showed that the tag is able to monitor the release of entrapped therapeutic agents. We found that liposomes release their cargo >100-fold faster in tissues in vivo than in conventional in vitro assays. This allowed us to design a liposomal formulation with enhanced ability to kill tumor associated macrophages. Our development opens up new opportunities for studying the chemical properties and pharmacodynamics of administered organic materials in an intact biological environment. This approach provides insight into the in vivo behavior of degradable materials, where the newly discovered information can guide the engineering of the next generation of imaging and therapeutic agents.

Activation of the transcription factor liver X receptor (LXR) has beneficial effects on macrophage lipid metabolism and inflammation, making it a potential candidate for therapeutic targeting in cardiometabolic disease. While small molecule delivery via nanomedicine has promising applications for a number of chronic diseases, questions remain as to how nanoparticle formulation might be tailored to suit different tissue microenvironments and aid in drug delivery. In the current study, we aimed to compare the in vitro drug delivering capability of three nanoparticle (NP) formulations encapsulating the LXR activator, GW-3965. We observed little difference in the base characteristics of standard PLGA-PEG NP when compared to two redox-active polymeric NP formulations, which we called redox-responsive (RR)1 and RR2. Moreover, we also observed similar uptake of these NP into primary mouse macrophages. We used the transcript and protein expression of the cholesterol efflux protein and LXR target ATP-binding cassette A1 (ABCA1) as a readout of GW-3956-induced LXR activation. Following an initial acute uptake period that was meant to mimic circulating exposure in vivo, we determined that although the induction of transcript expression was similar between NPs, treatment with the redox-sensitive RR1 NPs resulted in a higher level of ABCA1 protein. Our results suggest that NP formulations responsive to cellular cues may be an effective tool for targeted and disease-specific drug release.

Triple negative breast cancer (TNBC) accounts for the majority of breast cancer‐related deaths and remains the hardest breast cancer to treat due to the lack of specific therapeutic targets. While chemotherapy is the mainstay of systemic treatment for TNBC, it is associated with chemotherapy‐induced cancer stem cells (CSCs) and tumor regeneration. Here, it is found that Wnt and YAP target genes that have been closely associated with CSCs are highly expressed in TNBC patient tumors and negatively correlated with patient survival. Therefore, a nanotherapeutic strategy is employed, using nanomaterials that are approved by the FDA, and two co‐delivery nanoparticle platforms (NPs) are developed to target TNBC. These NPs contain Wnt inhibitor PRI‐724 (in clinical trials) and YAP/mevalonate inhibitor simvastatin (FDA‐approved). Toward clinical translation, nanotherapeutic efficacy is assessed in clinically relevant patient‐derived xenograft (PDX) models. These NPs in combination with the chemotherapeutic drug paclitaxel effectively halt the growth of both paclitaxel‐resistant and paclitaxel‐sensitive PDX tumors, and diminish the paclitaxel‐induced CSC enrichment around two to fourfold. Importantly, NPs also decrease the paclitaxel‐enhanced PDX tumorigenesis after secondary transplantation. Together, this study demonstrates the efficacy of two NP platforms using clinically translatable TNBC PDX models, suggesting their application potential for the treatment of TNBC.

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