PRECISE Scale: a quantitative classification for androgenetic alopecia and its application to hair transplantation.

BACKGROUND: Androgenetic alopecia (AGA) is a prevalent genetic condition that can affect both male and female, and is considered the most frequent form of hair loss. Traditional scales and methods of classifying AGA are basically qualitative. OBJECTIVE: This work aims to propose a quantitative scale to classify AGA in order to assist hair transplantation surgery. METHODS: Based on whole hairless and thinning areas that needs to receive follicular units in a hair transplantation procedure, basic equations to support the scale are proposed. Additionally, the study involves simulations that apply the classification system and compare its results with those of qualitative methods. RESULTS: The PRECISE scale utilizes a range of 0-10, using 30 cm2 as the measured standard of a bald area. For hair transplantation, 1500 follicular units (FU) are recommended for each score in the PRECISE scale. Technological and manual methods to measure the hairless and thinning areas are presented and discussed. This new quantitative classification, combined with different and complementary methods of measurement of hairless and thinning areas endorse the understanding of the clinical condition by the patient and the planning of a surgery procedure. CONCLUSION: The developed PRECISE scale brings a different way of classifying Androgenetic alopecia (AGA), through an essentially quantitative evaluation. It can be used to elaborate the best strategy for the hair transplantation surgery and to improve the outcomes. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these evidence-based medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Potential Use of Acrocomia aculeata Pulp Oil in the Modulation of Chronic Non-communicable Diseases, Especially in Diabetes: A Systematic Review.

INTRODUCTION: The fruit oil from Acrocomia aculeata (Macauba or Bocaiuva) is highly rich in antioxidants and other bioactive compounds, emerging as a natural source of high potential for the modulation of chronic non-communicable diseases (NCDs), like diabetes. Its effects on chronic NCDs are poorly studied yet. Our review aimed to evaluate the therapeutic results of pharmaceutical preparations containing Acrocomia aculeata pulp oil that are used for chronic NCDs. METHOD: A search was performed using PICO acronyms in English, Portuguese, and Spanish languages in the MEDLINE®, PubMed, EMBASE, Scopus, LILACs, and CENTRAL Cochrane Library databases. The degree of agreement for selection and eligibility was significant (Kappa= 0.992; 95% CI: 0.988-0.996). The difference between the intervention and control groups for blood glucose reduction was 63.5 ± 69.5 mg/dL (p<0.0001). RESULT: Overall, an improvement percentage of 55.1 ± 0.1 was observed for the variables associated with chronic NCDs, which represented 89.96% of the relative risk reduction (efficacy). CONCLUSION: The Acrocomia aculeate pulp oil exhibited promising results in experimental studies for glycemic control and reduction of a specific tumor, indicating a good potential to be explored for chronic NCDs treatment.

Advancements in Chitosan-Based Nanoparticles for Pulmonary Drug Delivery.

The evolution of respiratory diseases represents a considerable public health challenge, as they are among the leading causes of death worldwide. In this sense, in addition to the high prevalence of diseases such as asthma, chronic obstructive pulmonary disease, pneumonia, cystic fibrosis, and lung cancer, emerging respiratory diseases, particularly those caused by members of the coronavirus family, have contributed to a significant number of deaths on a global scale over the last two decades. Therefore, several studies have been conducted to optimize the efficacy of treatments against these diseases, focusing on pulmonary drug delivery using nanomedicine. Thus, the development of nanocarriers has emerged as a promising alternative to overcome the limitations of conventional therapy, by increasing drug bioavailability at the target site and reducing unwanted side effects. In this context, nanoparticles composed of chitosan (CS) show advantages over other nanocarriers because chitosan possesses intrinsic biological properties, such as anti-inflammatory, antimicrobial, and mucoadhesive capacity. Moreover, CS nanoparticles have the potential to enhance drug stability, prolong the duration of action, improve drug targeting, control drug release, optimize dissolution of poorly soluble drugs, and increase cell membrane permeability of hydrophobic drugs. These properties could optimize the performance of the drug after its pulmonary administration. Therefore, this review aims to discuss the potential of chitosan nanoparticles for pulmonary drug delivery, highlighting how their biological properties can improve the treatment of pulmonary diseases, including their synergistic action with the encapsulated drug.

The Influence of the Omicron Variant on RNA Extraction and RT-qPCR Detection of SARS-CoV-2 in a Laboratory in Brazil.

The emergence of SARS-CoV-2 variants can affect their detection via RT-qPCR. The Omicron variant has a greater affinity for the upper respiratory system and causes clinical conditions similar to bronchitis, as opposed to the pneumonitis-like conditions caused by other SARS-CoV-2 variants. This characteristic increases the viscosity of clinical samples collected for diagnosis. Coinciding with the arrival of the Omicron variant, we observed a failure in control gene expression in our laboratory. In this report, we have optimized a rapid nucleic acid extraction step to restore gene expression and detect the presence of the SARS-CoV-2 virus. We reevaluated 3000 samples, compared variant types occurring in different time periods, and confirmed that the presence of the Omicron variant was responsible for changes observed in the characteristics of these clinical samples. For samples with large amounts of mucus, such as those containing the Omicron variant, a modification to the nucleic acid extraction step was sufficient to restore the quality of RT-qPCR results.

Kaurenoic acid nanocarriers regulates cytokine production and inhibit breast cancer cell migration.

Breast cancer is the type of cancer with the highest incidence in women around the world. Noteworthy, the triple-negative subtype affects 20% of the patients while presenting the highest death rate among subtypes. This is due to its aggressive phenotype and the capability of invading other tissues. In general, tumor-associated macrophages (TAM) and other immune cells, are responsible for maintaining a favorable tumor microenvironment for inflammation and metastasis by secreting several mediators such as pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α, chemokines like CCL2, and other proteins, as metalloproteinases of matrix (MMP). On the other hand, immunomodulatory agents can interfere in the immune response of TAM and change the disease prognosis. In this work, we prepared nanostructured lipid carriers containing kaurenoic acid (NLC-KA) to evaluate the effect on cytokine production in vitro of bone marrow-derived macrophages (BMDM) and the migratory process of 4 T1 breast cancer cells. NLC-KA prepared from a blend of natural lipids was shown to have approximately 90 nm in diameter with low polydispersity index. To test the effect on cytokine production in vitro in NLC-KA treated BMDM, ELISA assay was performed and pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α were quantified. The formulation reduced the secretion of IL-1ß and TNF-α cytokines while presenting no hemolytic activity. Noteworthy, an anti-migratory effect in 4 T1 breast cancer cells treated with NLC-KA was observed in scratch assays. Further, MMP9 and CCL2 gene expressions in both BMDM and 4 T1 treated cells confirmed that the mechanism of inhibition of migration is related to the blockade of this pathway by KA. Finally, cell invasion assays confirmed that NLC-KA treatment resulted in less invasiveness of 4 T1 cells than control, and it is independent of CCL2 stimulus or BMDM direct stimulus. Ultimately, NLC-KA was able to regulate the cytokine production in vitro and reduce the migration of 4 T1 breast cancer cells by decreasing MMP9 gene expression.

Comparison of Rapid Nucleic Acid Extraction Methods for SARS-CoV-2 Detection by RT-qPCR.

Since 2020, humanity has been facing the COVID-19 pandemic, a respiratory disease caused by the SARS-CoV-2. The world's response to pandemic went through the development of diagnostics, vaccines and medicines. Regarding diagnostics, an enormous challenge was faced due to shortage of materials to collect and process the samples, and to perform reliable mass diagnosis by RT-qPCR. In particular, time-consuming and high cost of nucleic acid extraction procedures have hampered the diagnosis; moreover, several steps in the routine for the preparation of the material makes the extracted sample susceptible to contamination. Here two rapid nucleic acid extraction reagents were compared as extraction procedures for SARS-CoV-2 detection in clinical samples by singleplex and multiplex RT-qPCR analysis, using different transport media, samples with high and low viral load, and different PCR machines. As observed, rapid nucleic acid extraction procedures can be applied for reliable diagnosis using a TaqMan-based assay, over multiple platforms. Ultimately, prompt RNA extraction may reduce costs with reagents and plastics, the chances of contamination, and the overall time to diagnosis by RT-qPCR.

Nanostructured Lipid Carriers for the Formulation of Topical Anti-Inflammatory Nanomedicines Based on Natural Substances.

The main function of the skin is to protect the body from the external environment. However, the skin can undergo inflammatory processes, due to genetic, hormonal, or environmental factors. When the defense system is overloaded, there is an increase in pro-inflammatory mediators and reactive oxygen species (ROS), which results in skin disorders. Among the substances used to treat these inflammatory processes, many natural substances with anti-inflammatory and antioxidant properties are being studied: nature is yet an abundant source to obtain diverse pharmacological actives. The treatment of skin diseases is usually focused on topical application, as it reduces the risk of systemic side effects and prevents drug degradation by first-pass metabolism. Thus, the properties of drug delivery vehicles can facilitate or inhibit its permeation. Due to the hydrophobic nature of the skin, a promising strategy to improve dermal drug penetration is the use of lipid-based nanoparticles, such as nanostructured lipid carriers (NLC). Therefore, in this review, we present NLC as a tool to improve dermal administration of natural substances with anti-inflammatory properties.

Licochalcone A-loaded solid lipid nanoparticles improve antischistosomal activity in vitro and in vivo.

Aim: To isolate licochalcone A (LicoA) from licorice, prepare LicoA-loaded solid lipid nanoparticles (L-SLNs) and evaluate the L-SLNs in vitro and in vivo against Schistosoma mansoni. Materials & methods: LicoA was obtained by chromatographic fractionation and encapsulated in SLNs by a modified high shear homogenization method. Results: L-SLNs showed high encapsulation efficiency, with satisfactory particle size, polydispersity index and Zeta potential. Transmission electron microscopy revealed that L-SLNs were rounded and homogenously distributed. Toxicity studies revealed that SLNs decreased the hemolytic and cytotoxic properties of LicoA. Treatment with L-SLNs showed in vivo efficacy against S. mansoni. Conclusion: L-SLNs are efficient in reducing worm burden and SLNs may be a promising delivery system for LicoA to treat S. mansoni infections.

Neuroprotective Effect of siRNA Entrapped in Hyaluronic Acid-Coated Lipoplexes by Intravitreal Administration.

Since the possibility of silencing specific genes linked to retinal degeneration has become a reality with the use of small interfering RNAs (siRNAs), this technology has been widely studied to promote the treatment of several ocular diseases. Despite recent advances, the clinical success of gene silencing in the retina is significantly reduced by inherent anatomical and physiological ocular barriers, and new strategies are required to achieve intraocular therapeutic effectiveness. In this study, we developed lipoplexes, prepared with sodium alginate as an adjuvant and strategically coated with hyaluronic acid (HA-LIP), and investigated the potential neuroprotective effect of these systems in a retinal light damage model. Successful functionalization of the lipoplexes with hyaluronic acid was indicated in the dynamic light scattering and transmission electron microscopy results. Moreover, these HA-LIP nanoparticles were able to protect and deliver siRNA molecules targeting caspase-3 into the retina. After retinal degeneration induced by high light exposure, in vitro and in vivo quantitative reverse transcription-PCR (RT-qPCR) assays demonstrated significant inhibition of caspase-3 expression by HA-LIP. Furthermore, these systems were shown to be safe, as no evidence of retinal toxicity was observed by electroretinography, clinical evaluation or histology.

Short interfering RNA delivered by a hybrid nanoparticle targeting VEGF: Biodistribution and anti-tumor effect.

BACKGROUND: The use of RNA interference (iRNA) therapy has proved to be an interesting target therapy for the cancer treatment; however, siRNAs are unstable and quickly eliminated from the bloodstream. To face these barriers, the use of biocompatible and efficient nanocarriers emerges as an alternative to improve the success application of iRNA to the cancer, including breast cancer. RESULTS: A hybrid nanocarrier composed of calcium phosphate as the inorganic phase and a block copolymer containing polyanions as organic phase, named HNPs, was developed to deliver VEGF siRNA into metastatic breast cancer in mice. The particles presented a rounded shape by TEM images with average size measured by DLS suitable and biocompatible for biomedical applications. The XPS and EDS spectra confirmed the hybrid composition of the nanoparticles. Moreover, after intravenous administration, the particles accumulated mainly in the tumor site and kidneys, which demonstrates the tumor targeting accumulation through the Enhanced Permeability and Retention Effect (EPR). A significant decrease in size of the tumors treated with the nanoparticles containing siVEGF (HNPs-siVEGF) was observed and the reduction was related to enhanced tumor accumulation of siRNA as well as in vivo VEGF silencing at gene and protein levels. CONCLUSION: The hybrid system prepared was successful in promoting the RNAi effect in vivo with very low toxicity. GENERAL SIGNIFICANCE: This study shows the valuable development of a hybrid nanoparticle carrying VEGF siRNA, as well as their tumor targeting, accumulation and reduction in mice triple-negative breast cancer.

Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and quickly spread around the world, forcing global health authorities to develop protocols for its diagnosis. Here we report dimer formation in the N2 primers-probe set (CDC 2019-nCoV Real-Time RT-PCR) used in the diagnostic routine, and propose alternatives to reduce dimerization events. Late unspecific amplifications were visualized in 56.4% of negative samples and 57.1% of no-template control, but not in positive samples or positive control. In silico analysis and gel electrophoresis confirmed the dimer formation. The RT-qPCR parameters were optimized and the late unspecific amplifications decreased to 11.5% in negative samples and no-template control. The adjustment of PCR parameters was essential to reduce the risk of false-positives results and to avoid inclusive results requiring repeat testing, which increases the costs and generates delays in results or even unnecessary requests for new samples.

Improved anti-Cutibacterium acnes activity of tea tree oil-loaded chitosan-poly(ε-caprolactone) core-shell nanocapsules.

The purpose of this study was to develop tea tree oil (TTO)-loaded chitosan-poly(ε-caprolactone) core-shell nanocapsules (NC-TTO-Ch) aiming the topical acne treatment. TTO was analyzed by gas chromatography-mass spectrometry, and nanocapsules were characterized regarding mean particle size (Z-average), polydispersity index (PdI), zeta potential (ZP), pH, entrapment efficiency (EE), morphology by Atomic Force Microscopy (AFM), and anti-Cutibacterium acnes activity. The main constituents of TTO were terpinen-4-ol (37.11 %), γ-terpinene (16.32 %), α-terpinene (8.19 %), ρ-cimene (6.56 %), and α-terpineol (6.07 %). NC-TTO-Ch presented Z-average of 268.0 ± 3.8 nm and monodisperse size distribution (PdI < 0.3). After coating the nanocapsules with chitosan, we observed an inversion in ZP to a positive value (+31.0 ± 1.8 mV). This finding may indicate the presence of chitosan on the nanocapsules' surface, which was corroborated by the AFM images. In addition, NC-TTO-Ch showed a slightly acidic pH (∼5.0), compatible with topical application. The EE, based on Terpinen-4-ol concentration, was approximately 95 %. This data suggests the nanocapsules' ability to reduce the TTO volatilization. Furthermore, NC-TTO-Ch showed significant anti-C. acnes activity, with a 4× reduction in the minimum inhibitory concentration, compared to TTO and a decrease in C. acnes cell viability, with an increase in the percentage of dead cells (17 %) compared to growth control (6.6 %) and TTO (9.7 %). Therefore, chitosan-poly(ε-caprolactone) core-shell nanocapsules are a promising tool for TTO delivery, aiming at the activity against C. acnes for the topical acne treatment.

Multifunctional hybrid nanoparticles as magnetic delivery systems for siRNA targeting the HER2 gene in breast cancer cells.

Breast cancer is a major cause of death among women worldwide. Resistance to conventional therapies has been observed in HER2-positive breast cancer patients, indicating the need for more effective treatments. Small interfering RNA (siRNA) therapy is an attractive strategy against HER2-positive tumors, but its success depends largely on the efficient delivery of agents to target tissues. In this study, we prepared a magnetic hybrid nanostructure composed of iron oxide nanoparticles coated with caffeic acid and stabilized by layers of calcium phosphate and PEG-polyanion block copolymer for incorporation of siRNA. Transmission electron microscopy images showed monodisperse, neutrally charged compact spheres sized <100 nm. Dynamic light scattering and nanoparticle tracking analysis revealed that the nanostructure had an average hydrodynamic diameter of 130 nm. Nanoparticle suspensions remained stable over 42 days of storage at 4 and 25 °C. Unloaded caffeic acid-magnetic calcium phosphate (Caf-MCaP) nanoparticles were not cytotoxic, and loaded nanoparticles were successfully taken up by the HER2-positive breast cancer cell line HCC1954, even more so under magnetic guidance. Nanoparticles escaped endosomal degradation and delivered siRNA into the cytoplasm, inducing HER2 gene silencing.

Magnetically responsive hybrid nanoparticles for in vitro siRNA delivery to breast cancer cells.

Short interfering RNA (siRNA) showed to be a viable alternative to a better prognosis in cancer therapy. Nevertheless, the successful application of this strategy still depends on the development of nanocarriers for the safe delivery of siRNA into the diseased tissue, which mostly occurs by passive accumulation. When an external magnetic field is applied, magnetic nanoparticles biodistribution is partially modulated to favor accumulation in a target tissue. In this work we designed a novel magnetic responsive siRNA nanocarrier. The new delivery system is composed of superparamagnetic iron oxide nanoparticles (SPIONs) coated with calcium phosphate (CaP) and PEG-polyanion block copolymers, which are known to be biocompatible. The nanoparticles presented rounded shape with small size and narrow distribution suitable for biomedical applications. TEM images showed dark spheres in the core surrounded by a lower electron density material in the corona. The X-ray photoelectron spectra (XPS) confirmed CaP-polymer coating of the magnetic core. In addition, the coating procedure did not affect the superparamagnetic property as showed using a vibrating sample magnetometer (VSM). With a high loading efficiency (80%), the nanoparticles enhanced vascular endothelium growth factor (VEGF) silencing in breast cancer cells in vitro, at gene and protein levels (~60% and 40%, respectively), without associated toxicity. Iron and siRNA quantification showed that the novel nanoparticles move towards a magnetic source carrying siRNA molecules. Therefore, these novel nanoparticles are a promising tool for cancer therapy based on RNAi effect, added by a magnetic capability to further modulate siRNA accumulation in the target tissue.

Genetic, reproductive and oxidative damage in mice triggered by co-exposure of nanoparticles: From a hypothetical scenario to a real concern.

Humans are potentially exposed to multiple nanoparticles kinds through nanotechnology-based consumer products. There is insufficient data on the in vivo toxicity of nanotechnology products, as well as no data on the possible toxicity, including genotoxicity and reproductive toxicity of co-exposure to different kind of nanoparticles. In this work, solid lipid nanoparticles (SLNs) and superparamagnetic iron oxide nanoparticles (SPIONs) were selected for evaluation of a hypothetical condition of in vivo co-exposure. Genotoxicity of SPIONs and SLNs was performed separately and in 1:1 mixture in mice. Bone marrow micronucleus assay, sperm morphology test, and sperm count were carried out. Also, the serum ALT and AST activities; and hematological parameters of the treated mice were analyzed. The results showed a significant increase (p < 0.05) in micronucleated polychromatic erythrocytes (MNPCE) and nuclear abnormalities (NA) in SPIONs, SLNs and their mixture treated mice. The mixture induced the highest frequency of MNPCE and NA. A similar result was observed in the sperm morphology test, with the mixture inducing the highest sperm abnormalities, followed by SLNs and the least by SPIONs. Significant alteration to RDW, MCHC, MCV, GRAN, and platelets, as well as increased activities of serum AST were observed in the mice treated with a mixture of the two kinds of nanoparticles. Calculation of interaction factor showed a possible synergistic effect between SPIONs and SLNs in MNPCE, NA and sperm morphology studied. Even as a hypothetical scenario of co-exposure to SLNs and SPIONs, this study showed, for the first time, that co-exposure to SPIONs and SLNs is more genotoxic to somatic and germ cells than their individual exposure.

Knockdown of antiapoptotic genes in breast cancer cells by siRNA loaded into hybrid nanoparticles.

Tumorigenesis is related to an imbalance in controlling mechanisms of apoptosis. Expression of the genes BCL-2 and BCL-xL results in the promotion of cell survival by inhibiting apoptosis. Thus, a novel approach to suppress antiapoptotic genes is the use of small interfering RNA (siRNA) in cancer cells. However, there are some limitations for the application of siRNA such as the need for vectors to pass the cell membrane and deliver the nucleic acid. In this study CaP-siRNA-PEG-polyanion hybrid nanoparticles were developed to promote siRNA delivery to cultured human breast cancer cells (MCF-7) in order to evaluate whether the silencing of antiapoptotic genes BCL-2 and BCL-xL by siRNA would increase cancer cell death. After 48 h of incubation the expression of BCL-2 and BCL-xL genes decreased to 49% and 23%, respectively. The siRNA sequence used induced cancer cell death at a concentration of 200 nM siRNA after 72 h of incubation. As the targeted proteins are related to the resistance to chemotherapeutic drugs, the nanocarriers systems were also tested in the presence of doxorubicin (DOX). The results showed a significant reduction in the CC50 of the DOX, after silencing the antiapoptotic genes. In addition, an increase in apoptotic cell counts for both incubations conditions was observed as well. In conclusion, silencing antiapoptotic genes such as BCL-2 and BCL-xL through the use of siRNA carried by hybrid nanoparticles showed to be effective in vitro, and presents a promising strategy for pre-clinical analysis, especially when combined with DOX against breast cancer.

Influence of surfactant and lipid type on the physicochemical properties and biocompatibility of solid lipid nanoparticles.

Nine types of solid lipid nanoparticle (SLN) formulations were produced using tripalmitin (TPM), glyceryl monostearate (GM) or stearic acid (SA), stabilized with lecithin S75 and polysorbate 80. Formulations were prepared presenting PI values within 0.25 to 0.30, and the physicochemical properties, stability upon storage and biocompatibility were evaluated. The average particle size ranged from 116 to 306 nm, with a negative surface charge around -11 mV. SLN presented good stability up to 60 days. The SLN manufactured using SA could not be measured by DLS due to the reflective feature of this formulation. However, TEM images revealed that SA nanoparticles presented square/rod shapes with an approximate size of 100 nm. Regarding biocompatibility aspects, SA nanoparticles showed toxicity in fibroblasts, causing cell death, and produced high hemolytic rates, indicating toxicity to red blood cells. This finding might be related to lipid type, as well as, the shape of the nanoparticles. No morphological alterations and hemolytic effects were observed in cells incubated with SLN containing TPM and GM. The SLN containing TPM and GM showed long-term stability, suggesting good shelf-life. The results indicate high toxicity of SLN prepared with SA, and strongly suggest that the components of the formulation should be analyzed in combination rather than separately to avoid misinterpretation of the results.

Precise engineering of siRNA delivery vehicles to tumors using polyion complexes and gold nanoparticles.

For systemic delivery of siRNA to solid tumors, a size-regulated and reversibly stabilized nanoarchitecture was constructed by using a 20 kDa siRNA-loaded unimer polyion complex (uPIC) and 20 nm gold nanoparticle (AuNP). The uPIC was selectively prepared by charge-matched polyionic complexation of a poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) copolymer bearing ∼40 positive charges (and thiol group at the ω-end) with a single siRNA bearing 40 negative charges. The thiol group at the ω-end of PEG-PLL further enabled successful conjugation of the uPICs onto the single AuNP through coordinate bonding, generating a nanoarchitecture (uPIC-AuNP) with a size of 38 nm and a narrow size distribution. In contrast, mixing thiolated PEG-PLLs and AuNPs produced a large aggregate in the absence of siRNA, suggesting the essential role of the preformed uPIC in the formation of nanoarchitecture. The smart uPIC-AuNPs were stable in serum-containing media and more resistant against heparin-induced counter polyanion exchange, compared to uPICs alone. On the other hand, the treatment of uPIC-AuNPs with an intracellular concentration of glutathione substantially compromised their stability and triggered the release of siRNA, demonstrating the reversible stability of these nanoarchitectures relative to thiol exchange and negatively charged AuNP surface. The uPIC-AuNPs efficiently delivered siRNA into cultured cancer cells, facilitating significant sequence-specific gene silencing without cytotoxicity. Systemically administered uPIC-AuNPs showed appreciably longer blood circulation time compared to controls, i.e., bare AuNPs and uPICs, indicating that the conjugation of uPICs onto AuNP was crucial for enhancing blood circulation time. Finally, the uPIC-AuNPs efficiently accumulated in a subcutaneously inoculated luciferase-expressing cervical cancer (HeLa-Luc) model and achieved significant luciferase gene silencing in the tumor tissue. These results demonstrate the strong potential of uPIC-AuNP nanoarchitectures for systemic siRNA delivery to solid tumors.

Fine-tuning of charge-conversion polymer structure for efficient endosomal escape of siRNA-loaded calcium phosphate hybrid micelles.

For efficient delivery of siRNA into the cytoplasm, a smart block copolymer of poly(ethylene glycol) and charge-conversion polymer (PEG-CCP) is developed by introducing 2-propionic-3-methylmaleic (PMM) amide as an anionic protective group into side chains of an endosome-disrupting cationic polyaspartamide derivative. The PMM amide moiety is highly susceptible to acid hydrolysis, generating the parent cationic polyaspartamide derivative at endosomal acidic pH 5.5 more rapidly than a previously synthesized cis-aconitic (ACO) amide control. The PMM-based polymer is successfully integrated into a calcium phosphate (CaP) nanoparticle with siRNA, constructing PEGylated hybrid micelles (PMM micelles) having a sub-100 nm size at extracellular neutral pH 7.4. Ultimately, PMM micelles achieve the significantly higher gene silencing efficiency in cultured cancer cells, compared to ACO control micelles, probably due to the efficient endosomal escape of the PMM micelles. Thus, it is demonstrated that fine-tuning of acid-labile structures in CCP improves the delivery performance of siRNA-loaded nanocarriers.

Systemic siRNA delivery to a spontaneous pancreatic tumor model in transgenic mice by PEGylated calcium phosphate hybrid micelles.

Efficient systems for delivery of small interfering RNA (siRNA) are required for clinical application of RNA interference (RNAi) in cancer therapy. Herein, we developed a safe and efficient nanocarrier comprising poly(ethylene glycol)-block-charge-conversional polymer (PEG-CCP)/calcium phosphate (CaP) hybrid micelles for systemic delivery of siRNA and studied their efficacy in spontaneous bioluminescent pancreatic tumors from transgenic mice. PEG-CCP was engineered to provide the siRNA-loaded hybrid micelles with enhanced colloidal stability and biocompatibility due to the PEG capsule and with endosome-disrupting functionality due to the acidic pH-responsive CCP segment where the polyanionic structure could be converted to polycationic structure at acidic pH through cis-aconitic amide cleavage. The resulting hybrid micelles were confirmed to have a diameter of <50nm, with a narrow size distribution. Intravenously injected hybrid micelles significantly reduced the luciferase-based luminescent signal from the spontaneous pancreatic tumors in an siRNA sequence-specific manner. The gene silencing activity of the hybrid micelles correlated with their preferential tumor accumulation, as indicated by fluorescence imaging and histological analysis. Moreover, there were no significant changes in hematological parameters in mice treated with the hybrid micelles. These results demonstrate the great potential of the hybrid micelles as siRNA carriers for RNAi-based cancer therapy.