Delivery of miRNAs Using Porous Silicon Nanoparticles Incorporated into 3D Hydrogels Enhances MSC Osteogenesis by Modulation of Fatty Acid Signaling and Silicon Degradation.

Strategies incorporating mesenchymal stromal cells (MSC), hydrogels and osteoinductive signals offer promise for bone repair. Osteoinductive signals such as growth factors face challenges in clinical translation due to their high cost, low stability and immunogenicity leading to interest in microRNAs as a simple, inexpensive and powerful alternative. The selection of appropriate miRNA candidates and their efficient delivery must be optimised to make this a reality. This study evaluated pro-osteogenic miRNAs and used porous silicon nanoparticles modified with polyamidoamine dendrimers (PAMAM-pSiNP) to deliver these to MSC encapsulated within gelatin-PEG hydrogels. miR-29b-3p, miR-101-3p and miR-125b-5p are strongly pro-osteogenic and are shown to target FASN and ELOVL4 in the fatty acid biosynthesis pathway to modulate MSC osteogenesis. Hydrogel delivery of miRNA:PAMAM-pSiNP complexes enhanced transfection compared to 2D. The osteogenic potential of hBMSC in hydrogels with miR125b:PAMAM-pSiNP complexes is evaluated. Importantly, a dual-effect on osteogenesis occurred, with miRNAs increasing expression of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2) whilst the pSiNPs enhanced mineralisation, likely via degradation into silicic acid. Overall, this work presents insights into the role of miRNAs and fatty acid signalling in osteogenesis, providing future targets to improve bone formation and a promising system to enhance bone tissue engineering.

Combination of Chemotherapy and Mild Hyperthermia Using Targeted Nanoparticles: A Potential Treatment Modality for Breast Cancer.

Despite the clinical benefits that chemotherapeutics has had on the treatment of breast cancer, drug resistance remains one of the main obstacles to curative cancer therapy. Nanomedicines allow therapeutics to be more targeted and effective, resulting in enhanced treatment success, reduced side effects, and the possibility of minimising drug resistance by the co-delivery of therapeutic agents. Porous silicon nanoparticles (pSiNPs) have been established as efficient vectors for drug delivery. Their high surface area makes them an ideal carrier for the administration of multiple therapeutics, providing the means to apply multiple attacks to the tumour. Moreover, immobilising targeting ligands on the pSiNP surface helps direct them selectively to cancer cells, thereby reducing harm to normal tissues. Here, we engineered breast cancer-targeted pSiNPs co-loaded with an anticancer drug and gold nanoclusters (AuNCs). AuNCs have the capacity to induce hyperthermia when exposed to a radiofrequency field. Using monolayer and 3D cell cultures, we demonstrate that the cell-killing efficacy of combined hyperthermia and chemotherapy via targeted pSiNPs is 1.5-fold higher than applying monotherapy and 3.5-fold higher compared to using a nontargeted system with combined therapeutics. The results not only demonstrate targeted pSiNPs as a successful nanocarrier for combination therapy but also confirm it as a versatile platform with the potential to be used for personalised medicine.

Differential Surface Engineering Generates Core-Shell Porous Silicon Nanoparticles for Controlled and Targeted Delivery of an Anticancer Drug.

An approach to differentially modify the internal surface of porous silicon nanoparticles (pSiNPs) with hydrophobic dodecene and the external surface with antifouling poly-N-(2-hydroxypropyl) acrylamide (polyHPAm) as well as a cell-targeting peptide was developed. Specifically, to generate these core-shell pSiNPs, the interior surface of a porous silicon (pSi) film was hydrosilylated with 1-dodecene, followed by ultrasonication to create pSiNPs. The new external surfaces were modified by silanization with a polymerization initiator, and surface-initiated atom transfer radical polymerization was performed to introduce polyHPAm brushes. Afterward, a fraction of the polymer side chain hydroxyl groups was activated to conjugate cRGDfK─a peptide with a high affinity and selectivity for the ανß3 integrin receptor that is overexpressed in prostate and melanoma cancers. Finally, camptothecin, a hydrophobic anti-cancer drug, was successfully loaded into the pores. This drug delivery system showed excellent colloidal stability in a cell culture medium, and the in vitro drug release kinetics could be fine-tuned by the combination of internal and external surface modifications. In vitro studies by confocal microscopy and flow cytometry revealed improved cellular association attributed to cRGDfK. Furthermore, the cell viability results showed that the drug-loaded and peptide-functionalized nanoparticles had enhanced cytotoxicity toward a C4-2B prostate carcinoma cell line in both 2D cell culture and a 3D spheroid model.

Porous Silicon Nanocarriers with Stimulus-Cleavable Linkers for Effective Cancer Therapy.

Porous silicon nanoparticles (pSiNPs) are widely utilized as drug carriers due to their excellent biocompatibility, large surface area, and versatile surface chemistry. However, the dispersion in pore size and biodegradability of pSiNPs arguably have hindered the application of pSiNPs for controlled drug release. Here, a step-changing solution to this problem is described involving the design, synthesis, and application of three different linker-drug conjugates comprising anticancer drug doxorubicin (DOX) and different stimulus-cleavable linkers (SCLs) including the photocleavable linker (ortho-nitrobenzyl), pH-cleavable linker (hydrazone), and enzyme-cleavable linker (ß-glucuronide). These SCL-DOX conjugates are covalently attached to the surface of pSiNP via copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC, i.e., click reaction) to afford pSiNP-SCL-DOXs. The mass loading of the covalent conjugation approach for pSiNP-SCL-DOX reaches over 250 µg of DOX per mg of pSiNPs, which is notably twice the mass loading achieved by noncovalent loading. Moreover, the covalent conjugation between SCL-DOX and pSiNPs endows the pSiNPs with excellent stability and highly controlled release behavior. When tested in both in vitro and in vivo tumor models, the pSiNP-SCL-DOXs induces excellent tumor growth inhibition.

ELOVL5 Is a Critical and Targetable Fatty Acid Elongase in Prostate Cancer.

The androgen receptor (AR) is the key oncogenic driver of prostate cancer, and despite implementation of novel AR targeting therapies, outcomes for metastatic disease remain dismal. There is an urgent need to better understand androgen-regulated cellular processes to more effectively target the AR dependence of prostate cancer cells through new therapeutic vulnerabilities. Transcriptomic studies have consistently identified lipid metabolism as a hallmark of enhanced AR signaling in prostate cancer, yet the relationship between AR and the lipidome remains undefined. Using mass spectrometry-based lipidomics, this study reveals increased fatty acyl chain length in phospholipids from prostate cancer cells and patient-derived explants as one of the most striking androgen-regulated changes to lipid metabolism. Potent and direct AR-mediated induction of ELOVL fatty acid elongase 5 (ELOVL5), an enzyme that catalyzes fatty acid elongation, was demonstrated in prostate cancer cells, xenografts, and clinical tumors. Assessment of mRNA and protein in large-scale data sets revealed ELOVL5 as the predominant ELOVL expressed and upregulated in prostate cancer compared with nonmalignant prostate. ELOVL5 depletion markedly altered mitochondrial morphology and function, leading to excess generation of reactive oxygen species and resulting in suppression of prostate cancer cell proliferation, 3D growth, and in vivo tumor growth and metastasis. Supplementation with the monounsaturated fatty acid cis-vaccenic acid, a direct product of ELOVL5 elongation, reversed the oxidative stress and associated cell proliferation and migration effects of ELOVL5 knockdown. Collectively, these results identify lipid elongation as a protumorigenic metabolic pathway in prostate cancer that is androgen-regulated, critical for metastasis, and targetable via ELOVL5. SIGNIFICANCE: This study identifies phospholipid elongation as a new metabolic target of androgen action that is critical for prostate tumor metastasis.

Engineering Fluorescent Gold Nanoclusters Using Xanthate-Functionalized Hydrophilic Polymers: Toward Enhanced Monodispersity and Stability.

We introduce xanthate-functionalized poly(cyclic imino ethers)s (PCIEs), specifically poly(2-ethyl-2-oxazoline) and poly(2-ethyl-2-oxazine) given their stealth characteristics, as an attractive alternative to conventional thiol-based ligands for the synthesis of highly monodisperse and fluorescent gold nanoclusters (AuNCs). The xanthate in the PCIEs interacts with Au ions, acting as a well-controlled template for the direct formation of PCIE-AuNCs. This method yields red-emitting AuNCs with a narrow emission peak (λem = 645 nm), good quantum yield (4.3-4.8%), long fluorescence decay time (∼722-844 ns), and unprecedented product yield (>98%). The PCIE-AuNCs exhibit long-term colloidal stability, biocompatibility, and antifouling properties, enabling a prolonged blood circulation, lower nonspecific accumulation in major organs, and better renal clearance when compared with AuNCs without polymer coating. The advances made here in the synthesis of metal nanoclusters using xanthate-functionalized PCIEs could propel the production of highly monodisperse, biocompatible, and renally clearable nanoprobes in large-scale for different theranostic applications.

Nanobody-displaying porous silicon nanoparticles for the co-delivery of siRNA and doxorubicin.

Targeted delivery of chemotherapeutics to cancer cells has the potential to yield high drug concentrations in cancer cells while minimizing any unwanted side effects. However, the development of multidrug resistance in cancer cells may impede the accumulation of chemotherapy drugs within these, decreasing its therapeutic efficacy. Downregulation of multidrug resistance-related proteins such as MRP1 with small interfering RNA (siRNA) is a promising approach in the reversal of drug resistance. The co-delivery of doxorubicin (Dox) and siRNA against MRP1 (siMRP1) by using nanoparticles comprised of biocompatible porous silicon (pSi) presents itself as a novel opportunity to utilize the biomaterial's high loading capacity and large accessible surface area. Additionally, to increase the selectivity and retention of the delivery vehicle at the tumor site, nanobodies were incorporated onto the nanoparticle surface via a polyethylene glycol (PEG) linker directed towards either the epidermal growth factor receptor (EGFR) or the prostate specific membrane antigen (PSMA). The nanobody-displaying pSi nanoparticles (pSiNPs) demonstrated effective gene silencing, inhibiting MRP1 expression by 74 ± 6% and 74 ± 4% when incubated with EGFR-pSiNPs and PSMA-pSiNPs, respectively, in prostate cancer cells. The downregulation of MRP1 led to a further increase in cytotoxicity when both siRNA and Dox were delivered in conjunction in both cancer cell monocultures and spheroids when compared to free Dox or Dox and a scrambled sequence of siRNA. Altogether, nanobody-displaying pSiNPs are an effective carrier for the dual delivery of both siRNA and Dox for cancer treatment.

Patient-Derived Prostate Cancer Explants: A Clinically Relevant Model to Assess siRNA-Based Nanomedicines.

Over the last thirty years, research in nanomedicine has widely been focused on applications in cancer therapeutics. However, despite the plethora of reported nanoscale drug delivery systems that can successfully eradicate solid tumor xenografts in vivo, many of these formulations have not yet achieved clinical translation. This issue particularly pertains to the delivery of small interfering RNA (siRNA), a highly attractive tool for selective gene targeting. One of the likely reasons behind the lack of translation is that current in vivo models fail to recapitulate critical elements of clinical solid tumors that may influence drug response, such as cellular heterogeneity in the tumor microenvironment. This study incorporates a more clinically relevant model for assessing siRNA delivery systems; ex vivo culture of prostate cancer harvested from patients who have undergone radical prostatectomy, denoted patient-derived explants (PDE). The model retains native human tissue architecture, microenvironment, and cell signaling pathways. Porous silicon nanoparticles (pSiNPs) behavior in this model is investigated and compared with commonly used 3D cancer cell spheroids for their efficacy in the delivery of siRNA directed against the androgen receptor (AR), a key driver of prostate cancer.

Maximizing RNA Loading for Gene Silencing Using Porous Silicon Nanoparticles.

Gene silencing by RNA interference is a powerful technology with broad applications. However, this technology has been hampered by the instability of small interfering RNA (siRNA) molecules in physiological conditions and their inefficient delivery into the cytoplasm of target cells. Porous silicon nanoparticles have emerged as a potential delivery vehicle to overcome these limitations-being able to encapsulate RNA molecules within the porous matrix and protect them from degradation. Here, key variables were investigated that influence siRNA loading into porous silicon nanoparticles. The effect of modifying the surface of porous silicon nanoparticles with various amino-functional molecules as well as the effects of salt and chaotropic agents in facilitating siRNA loading was examined. Maximum siRNA loading of 413 µg/(mg of porous silicon nanoparticles) was found when the nanoparticles were modified by a fourth generation polyamidoamine dendrimer. Low concentrations of urea or salt increased loading capacity: an increase in RNA loading by 19% at a concentration of 0.05 M NaCl or 21% at a concentration of 0.25 M urea was observed when compared to loading in water. Lastly, it was demonstrated that dendrimer-functionalized nanocarriers are able to deliver siRNA against ELOVL5, a target for the treatment of advanced prostate cancer.