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As transporters of RNAi therapeutics in preclinical and clinical studies, the application of nanoparticles is often hindered by their susceptibility to opsonin-mediated clearance, poor biological stability, ineffectual targeting, and undesirable effects on healthy cells. Prolonging the blood circulation time while minimizing the off-target distribution and associated toxicity is indispensable for the establishment of a clinically viable delivery system for therapeutic small interfering RNAs (siRNAs). Herein, we report a scalable and straightforward approach to fabricate non-toxic and biodegradable pH-responsive strontium sulfite nanoparticles (SSNs) wrapped with a hydrophilic coating material, biotinylated PEG to lessen unforeseen biological interactions. Surface functionalization of SSNs with PEG led to the generation of small and uniformly distributed particles with a significant affinity towards siRNAs and augmented internalization into breast cancer cells. A triple quadrupole liquid chromatography-mass spectrometry (LC-MS) was deployed to identify the proteins entrapped onto the SSNs, with the help of SwissProt.Mus_musculus database. The results demonstrated the reduction of opsonin proteins adsorption owing to the stealth effect of PEG. The distribution of PEGylated SSNs in mice after 4 h and 24 h of intravenous administration in breast tumour-bearing mice was found to be significantly less to the organs of the reticuloendothelial system (RES) and augmented accumulation in the tumour region. The anti-EGFR siRNA-loaded PEG-SSNs exerted a significant inhibitory effect on tumour development in the murine breast cancer model without any significant toxicity to healthy tissues. Therefore, PEGylated SSNs open up a new avenue for tumour-selective efficient delivery of siRNAs in managing breast cancer.

The journey of chemotherapeutic drugs from the site of administration to the site of action is confronted by several factors including low bioavailability, uneven distribution in major organs, limited accessibility of drug molecules to the distant tumor tissues, and lower therapeutic indexes. These unavoidable features of classical chemotherapeutics necessitate an additional high, repetitive dose of drugs to obtain maximum therapeutic responses with the result of unintended adverse side effects. An erratic tumor microenvironment, notable drawbacks of conventional chemotherapy, and multidrug-resistant mechanisms of breast cancer cells warrant precisely designed therapeutics for the treatment of cancers. In recent decades, nanoparticles have been deployed for the delivery of standard anticancer drugs to maximize the therapeutic potency while minimizing the adverse effects to increase the quality and span of life. Several organic and inorganic nanoplatforms that have been designed exploiting the distinctive features of the tumor microenvironment and tumor cells offer favorable physicochemical properties and pharmacokinetic profiles of a parent drug, with delivery of higher amounts of the drug to the pathological site and its controlled release, thereby improving the balance between its efficacy and toxicity. Advances to this front have included design and construction of targeted nanoparticles by conjugating homing devices like peptide, ligand, and Fab on the surface of nanomaterials to navigate nanoparticledrug complexes towards the target tumor cell with minimal destruction of healthy cells. Furthermore, actively targeting nanoparticles can facilitate the delivery and cellular uptake of nanoparticle-loaded drug constructs via binding with specific receptors expressed aberrantly on the surface of a tumor cell. Herein, we present an overview of the principle of targeted delivery approaches, exploiting drug-nanoparticle conjugates with multiple targeting moieties to target specific receptors of breast cancer cells and highlighting therapeutic evaluation in preclinical studies. We conclude that an understanding of the translational gap and challenges would show the possible future directions to foster the development of novel targeted nanotherapeutics.

Oral delivery is considered as the most preferred and yet most challenging mode of drug administration; especially a fragile and sensitive peptide like insulin that shows extremely low bioavailability through the gastro-intestinal (GIT) route. To address this problem, we have designed a novel drug delivery system (DDS) using precipitation-induced Barium (Ba) salt particles. The DDS can load insulin molecules and transport them through the GIT route. There were several in vitro simulation tests carried out to prove the efficiency of Ba salt particles as oral delivery candidates. All three Ba salt particles (BaSO4, BaSO3, and BaCO3) showed very good loading of insulin (>70% in all formulations) and a degree of resistance throughout a wide range of pHs from basic to acidic conditions when assessed by spectrophotometry. Particles and insulin-associated particles were morphologically assessed and characterized using FE-SEM and FT-IR. A set of tests were designed and carried out with mucin to predict whether the particles are potentially capable of overcoming one of the barriers for crossing intestinal epithelium. The mucin binding experiment demonstrated 60–100% of mucin adhesion to the three different particles. FT-IR identifies the characteristic peaks for mucin protein, particles, and particle-mucin complex re-confirming mucin adhesion to the particles. Finally, the effectiveness of nano-insulin was tested on streptozotocin (STZ) induced diabetic rats. A short acting human insulin analog, insulin aspart, was loaded into Ba salt particles at a dose of 100 IU/Kg prior to oral administration. Among the three formulations, insulin aspart-loaded BaSO4 and BaCO3 particles dramatically reduced the existing hyperglycemia. BaSO4 with loaded Insulin showed an onset of glucose-lowering action within 1 hr, with blood glucose level measured significantly lower compared to the 2nd and 3rd h (p < 0.05). Insulin-loaded BaCO3 particles showed a significant decrease in blood glucose level at 1–2 h, although the glucose level started to show a slight rise at 3rd h and by 4th h, it was back to baseline level. However, although BaSO3 particles with loaded insulin showed a trend of reduction in blood glucose level, the reduction was not found to be significant (p < 0.05) at any point in time. Therefore, oral formulations of insulin/BaSO4 and insulin/BaCO3 particles were observed as effective as native insulin aspart subcutaneous formulation in terms of onset and duration of action. Further investigation will be needed to reveal bioavailability and mechanism of action of this novel Nano-Insulin formulations.

Breast cancer is the abnormal, uncontrollable proliferation of cells in the breast. Conventional treatment modalities like chemotherapy induce deteriorating side effects on healthy cells. Non-viral inorganic nanoparticles (NPs) confer exclusive characteristics, such as, stability, controllable shape and size, facile surface modification, and unique magnetic and optical properties which make them attractive drug carriers. Among them, carbonate apatite (CA) particles are pH-responsive in nature, enabling rapid intracellular drug release, but are typically heterogeneous with the tendency to self-aggregate. Here, we modified the nano-carrier by partially substituting Ca2+ with Mg2+ and Fe3+ into a basic lattice structure of CA, forming Fe/Mg-carbonate apatite (Fe/Mg-CA) NPs with the ability to mitigate self-aggregation, form unique protein corona in the presence of serum and efficiently deliver doxorubicin (DOX), an anti-cancer drug into breast cancer cells. Two formulations of Fe/Mg-CA NPs were generated by adding different concentrations of Fe3+ and Mg2+ along with a fixed amount of Ca2+ in bicarbonate buffered DMEM (Dulbecco’s Modified Eagle’s Medium), followed by 30 min incubation at 37 °C. Particles were characterized by turbidity analysis, z-average diameter and zeta potential measurement, optical microscopy, field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray (EDX), flame atomic absorption spectroscopy (FAAS), pH dissolution, drug binding, cellular uptake, thiazolyl blue tetrazolium bromide (MTT) assay, stability analysis, and protein corona study by LCMS (Liquid chromatography-mass spectrometry). Both formulations of Fe/Mg-CA displayed mostly uniform nano-sized particles with less tendency to aggregate. The EDX and FAAS elemental analysis confirmed the weight (%) of Ca, Fe and Mg, along with their Ca/P ratio in the particles. A constant drug binding efficiency was noticed with 5 μM to 10 μM of initial DOX concentration. A pH dissolution study of Fe/Mg-CA NPs revealed the quick release of DOX in acidic pH. Enhancement of cytotoxicity for the chemotherapy drug was greater for Fe/Mg-CA NPs as compared to CA NPs, which could be explained by an increase in cellular internalization as a result of the small z-average diameter of the former. The protein corona study by LCMS demonstrated that Fe/Mg-CA NPs exhibited the highest affinity towards transport proteins without binding with opsonins. Biodistribution study was performed to study the effect of DOX-loaded Fe/Mg-CA NPs on the tissue distribution of DOX in Balb/c 4T1 tumor-bearing mice. Both formulations of Fe/Mg-CA NPs have significantly increased the accumulation of DOX in tumors. Interestingly, high Fe/Mg-CA NPs exhibited less off-target distribution compared to low Fe/Mg-CA NPs. Furthermore, the blood plasma analysis revealed prolonged blood circulation half-life of DOX-loaded low and high Fe/Mg-CA NPs compared to free DOX solution. Modifying CA NPs with Fe3+ and Mg2+, thereby, led to the generation of nano-sized particles with less tendency to aggregate, enhancing the drug binding efficiency, cellular uptake, and cytotoxicity without hampering drug release in acidic pH, while improving the circulation half-life and tumor accumulation of DOX. Therefore, Fe/Mg-CA which predominantly forms a transport protein-related protein corona could be a proficient carrier for therapeutic delivery in breast cancer.

Background: The efficacy of chemotherapy is undermined by adverse side effects and chemoresistance of target tissues. Developing a drug delivery system can reduce off-target side effects and increase the efficacy of drugs by increasing their accumulation in target tissues. Inorganic salts have several advantages over other drug delivery vectors in that they are non-carcinogenic and less immunogenic than viral vectors and have a higher loading capacity and better controlled release than lipid and polymer vectors. Methods: MgF2 crystals were fabricated by mixing 20 mM MgCl2 and 10 mM NaF and incubating for 30 min at 37 °C. The crystals were characterized by absorbance, dynamic light scattering, microscopic observance, pH sensitivity test, SEM, EDX and FTIR. The binding efficacy to doxorubicin was assessed by measuring fluorescence intensity. pH-dependent doxorubicin release profile was used to assess the controlled release capability of the particle-drug complex. Cellular uptake was assessed by fluorescence microscopy. Cytotoxicity of the particles and the drug-particle complex were assessed using MTT assay to measure cell viability of MCF-7 cells. Results and Discussion: Particle size on average was estimated to be 2 nanocrystals were safe in lower concentrations, and when bound to doxorubicin, enhanced its uptake. The protein corona formed around MgF2 nanoparticles lacks typical opsonins but contains some dysopsonins. Conclusion: A drug delivery vector in the form of MgF2 nanocrystals has been developed to transport doxorubicin into breast cancer cells. It is pH-sensitive (allowing for controlled release), size-modifiable, simple and cheap to produce.

Inorganic nanoparticles hold great potential in the area of precision medicine, particularly for treating cancer owing to their unique physicochemical properties, biocompatibility and improved pharmacokinetics properties compared to their organic counterparts. Here we introduce strontium sulfite nanoparticles as new pH-responsive inorganic nanocarriers for efficient transport of siRNAs into breast cancer cells. We employed the simplest nanoprecipitation method to generate the strontium sulfite nanoparticles (SSNs) and demonstrated the dramatic roles of NaCl and d-glucose in particle growth stabilization in order to produce even smaller nanosize particles (Na-Glc-SSN) with high affinity towards negatively charged siRNA, enabling it to efficiently enter the cancer cells. Moreover, the nanoparticles were found to be degraded with a small drop in pH, suggesting their potential capability to undergo rapid dissolution at endosomal pH so as to release the payload. While these particles were found to be nontoxic to the cells, they showed higher potency in facilitating cancer cell death through intracellular delivery and release of oncogene-specific siRNAs targeting ros1 and egfr1 mRNA transcripts, than the strontium sulfite particles prepared in absence of NaCl and d-glucose, as confirmed by growth inhibition assay. The mouse plasma binding analysis by Q-TOF LC-MS/MS demonstrated less protein binding to smaller particles of Na-Glc-SSNs. The biodistribution studies of the particles after 4 h of treatment showed Na-Glc-SSNs had less off-target distribution than SSNs, and after 24 h, all siRNAs were cleared from all major organs except the tumors. ROS1 siRNA with its potential therapeutic role in treating 4T1-induced breast tumor was selected for subsequent in vivo tumor regression study, revealing that ROS1 siRNA-loaded SSNs exerted more significant anti-tumor effects than Na-Glc-SSNs carrying the same siRNA following intravenous administration, without any systemic toxicity. Thus, strontium sulfite emerged as a powerful siRNA delivery tool with potential applications in cancer gene therapy.

Specific gene knockdown facilitated by short interfering RNA (siRNA) is a potential approach for suppressing the expression of ion channels and transporter proteins to kill breast cancer cells. The overexpression of calcium ion channels and transporter genes is seen in the MCF-7 breast cancer cell line. Since naked siRNA is anionic and prone to nuclease-mediated degradation, it has limited permeability across the cationic cell membrane and short systemic half-life, respectively. Carbonate apatite (CA) nanoparticles were formulated, characterized, loaded with a series of siRNAs, and delivered into MCF-7 and 4T1 breast cancer cells to selectively knockdown the respective calcium and magnesium ion channels and transporters. Individual knockdown of TRPC6, TRPM7, TRPM8, SLC41A1, SLC41A2, ORAI1, ORAI3, and ATP2C1 genes showed significant reduction (p < 0.001) in cell viability depending on the cancer cell type. From a variety of combinations of siRNAs, the combination of TRPC6, TRPM8, SLC41A2, and MAGT1 siRNAs delivered via CA produced the greatest cell viability reduction, resulting in a cytotoxicity effect of 57.06 ± 3.72% (p < 0.05) and 59.83 ± 2.309% (p = 0.09) in 4T1 and MCF-7 cell lines, respectively. Some of the combinations were shown to suppress the Akt pathway in Western Blot analysis when compared to the controls. Therefore, CA-siRNA-facilitated gene knockdown in vitro holds a high prospect for deregulating cell proliferation and survival pathways through the modulation of Ca2+ signaling in breast cancer cells.

RNA Interference (RNAi) has brought revolutionary transformations in cancer management in the past two decades. RNAi-based therapeutics including siRNA and shRNA have immense scope to silence the expression of mutant cancer genes specifically in a therapeutic context. Although tremendous progress has been made to establish catalytic RNA as a new class of biologics for cancer management, a lot of extracellular and intracellular barriers still pose a long-lasting challenge on the way to clinical approval. A series of chemically suitable, safe and effective viral and non-viral carriers have emerged to overcome physiological barriers and ensure targeted delivery of RNAi. The newly invented carriers, delivery techniques and gene editing technology made current treatment protocols stronger to fight cancer. This review has provided a platform about the chronicle of siRNA development and challenges of RNAi therapeutics for laboratory to bedside translation focusing on recent advancement in siRNA delivery vehicles with their limitations. Furthermore, an overview of several animal model studies of siRNA- or shRNA-based cancer gene therapy over the past 15 years has been presented, highlighting the roles of genes in multiple cancers, pharmacokinetic parameters and critical evaluation. The review concludes with a future direction for the development of catalytic RNA vehicles and design strategies to make RNAi-based cancer gene therapy more promising to surmount cancer gene delivery challenges.