Targeted treatment of triple-negative-breast cancer through pH-triggered tumour associated macrophages using smart theranostic nanoformulations.

Triple-negative breast cancer (TNBC) represents 15-25 % of the new breast cancer cases diagnosed worldwide every year. TNBC is among the most aggressive and worst prognosis breast cancer, mainly because targeted therapies are not available. Herein, we developed a magnetic theranostic hybrid nanovehicle for targeted treatment of TNBC through pH-triggered tumour associated macrophages (TAMs) targeting. The lipid core of the nanovehicle was composed of a Carnaúba wax matrix that simultaneously incorporated iron oxide nanoparticles and doxorubicin (DOX) - a chemotherapeutic drug. These drug-loaded wax nanovehicles were modified with a combination of two functional and complementary molecules: (i) a mannose ligand (macrophage targeting) and (ii) an acid-sensitive sheddable polyethylene glycol (PEG) moiety (specificity). The TAMs targeting strategy relied on the mannose - mannose receptor recognition exclusively after acid-sensitive "shedding" of the PEG in the relatively low tumour microenvironment pH. The pH-induced targeting capability towards TAMs was confirmed in vitro in a J774A.1 macrophage cell line at different pH (7.4 and 6.5). Biocompatibility and efficacy of the final targeted formulations were demonstrated in vitro in the TNBC MDA-MB-231 cell line and in vivo in an M-Wnt tumour-bearing (TNBC) mouse model. A preferential accumulation of the DOX-loaded lipid nanovehicles in the tumours of M-Wnt-tumour bearing mice was observed, which resulted both on an efficient tumour growth inhibition and a significantly reduced off-target toxicity compared to free DOX. Additionally, the developed magnetic hybrid nanovehicles showed outstanding performances as T2-contrast agents in magnetic resonance imaging (r2 ≈ 400-600 mM-1·s-1) and as heat generating sources in magnetic hyperthermia (specific absorption rate, SAR ≈ 178 W·g-1Fe). These targeted magnetic hybrid nanovehicles emerge as a suitable theranostic option that responds to the urgent demand for more precise and personalized treatments, not only because they are able to offer localized imaging and therapeutic potential, but also because they allow to efficiently control the balance between safety and efficacy.

A solid lipid nanoparticle formulation of 4-(N)-docosahexaenoyl 2', 2'-difluorodeoxycytidine with increased solubility, stability, and antitumor activity.

Previously, we synthesized 4-(N)-docosahexaenoyl 2', 2'-difluorodeoxycytidine (DHA-dFdC), a novel lipophilic compound with a potent, broad-spectrum antitumor activity. Herein, we report a solid lipid nanoparticle (SLN) formulation of DHA-dFdC with improved apparent aqueous solubility, chemical stability, as well as efficacy in a mouse model. The SLNs were prepared from lecithin/glycerol monostearate-in-water emulsions emulsified with D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and Tween 20. The resultant DHA-dFdC-SLNs were 102.2 ±â€¯7.3 nm in diameter and increased the apparent solubility of DHA-dFdC in water to at least 5.2 mg/mL, more than 200-fold higher than its intrinsic water solubility. DHA-dFdC in a lyophilized powder of DHA-dFdC-SLNs was significantly more stable than the waxy solid of pure DHA-dFdC. DHA-dFdC-SLNs also showed an increased cytotoxicity against certain tumor cells than DHA-dFdC. The plasma concentration of DHA-dFdC in mice intravenously injected with DHA-dFdC-SLNs in dispersion followed a bi-exponential model, with a half-life of ~44 h. In mice bearing B16-F10 murine melanoma, DHA-dFdC-SLNs were significantly more effective than DHA-dFdC in controlling the tumor growth. In addition, histology evaluation revealed a high level of apoptosis and tumor encapsulation in tumors in mice treated with DHA-dFdC-SLNs. DHA-dFdC-SLNs represents a new DHA-dFdC formulation with improved antitumor activity.

Intranasal immunization with aluminum salt-adjuvanted dry powder vaccine.

Intranasal vaccination using dry powder vaccine formulation represents an attractive, non-invasive vaccination modality with better storage stability and added protection at the mucosal surfaces. Herein we report that it is feasible to induce specific mucosal and systemic antibody responses by intranasal immunization with a dry powder vaccine adjuvanted with an insoluble aluminum salt. The dry powder vaccine was prepared by thin-film freeze-drying of a model antigen, ovalbumin, adsorbed on aluminum (oxy)hydroxide as an adjuvant. Special emphasis was placed on the characterization of the dry powder vaccine formulation that can be realistically used in humans by a nasal dry powder delivery device. The vaccine powder was found to have "passable" to "good" flow properties, and the vaccine was uniformly distributed in the dry powder. An in vitro nasal deposition study using nasal casts of adult humans showed that around 90% of the powder was deposited in the nasal cavity. Intranasal immunization of rats with the dry powder vaccine elicited a specific serum antibody response as well as specific IgA responses in the nose and lung secretions of the rats. This study demonstrates the generation of systemic and mucosal immune responses by intranasal immunization using a dry powder vaccine adjuvanted with an aluminum salt.

Lipid nanoparticles with minimum burst release of TNF-α siRNA show strong activity against rheumatoid arthritis unresponsive to methotrexate.

TNF-α siRNA has shown promising therapeutic benefits in animal models of rheumatoid arthritis. However, there continues to be a need for siRNA delivery systems that have high siRNA encapsulation efficiency and minimum burst release of TNF-α siRNA, and can target inflamed tissues after intravenous administration. Herein we report a novel acid-sensitive sheddable PEGylated solid-lipid nanoparticle formulation of TNF-α-siRNA, AS-TNF-α-siRNA-SLNs, prepared by incorporating lipophilized TNF-α-siRNA into solid-lipid nanoparticles composed of biocompatible lipids such as lecithin and cholesterol. The nanoparticles are approximately 120 nm in diameter, have a high siRNA encapsulation efficiency (>90%) and a minimum burst release of siRNA (<5%), and increase the deilvery of the siRNA in chronic inflammation sites in mouse models, including in a mouse model with collagen-induced arthritis. Importantly, in a mouse model of collagen antibody-induced arthritis that does not respond to methotrexate therapy, intravenous injection of the AS-TNF-α-siRNA-SLNs significantly reduced paw thickness, bone loss, and histopathological scores. These findings highlight the potential of using this novel siRNA nanoparticle formulation to effectively treat arthritis, potentially in patients who do not respond adequately to methotrexate.

Uric acid and the vaccine adjuvant activity of aluminium (oxy)hydroxide nanoparticles.

In an effort to improve the adjuvanticity of insoluble aluminium salts, we discovered that the adjuvant activity of aluminium salt nanoparticles is significantly stronger than aluminium salt microparticles, likely related to nanoparticle's stronger ability to directly activate NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome as the nanoparticles are more efficiently taken up by phagocytic cells. Endogenous signals such as uric acid from cell damage or death caused by the cytotoxicity of aluminium salts are thought to indirectly activate inflammasome, prompting us to hypothesise that the potent adjuvant activity of aluminium salt nanoparticles is also related to their ability to stimulate uric acid production. In the present study, we prepared aluminium (oxy)hydroxide nanoparticles (∼ 30-100 nm) and microparticles (X50, 9.43 µm) and showed that intraperitoneal injection of mice with the nanoparticles, absorbed with ovalbumin, led to a significant increase in uric acid level in the peritoneal lavage, whereas the microparticles did not. The aluminium (oxy)hydroxide nanoparticles' ability to stimulate uric acid production was also confirmed in cell culture. We concluded that the stronger adjuvant activity of insoluble aluminium (oxy)hydroxide nanoparticles, relative to microparticles, may be attributed at least in part to their stronger ability to induce endogenous danger signals such as uric acid.

Methods to Prepare Aluminum Salt-Adjuvanted Vaccines.

Many human vaccines contain certain insoluble aluminum salts such as aluminum oxyhydroxide and aluminum hydroxyphosphate as vaccine adjuvants to boost the immunogenicity of the vaccines. Aluminum salts have been used as vaccine adjuvants for decades and have an established, favorable safety profile. However, preparing aluminum salts and aluminum salt-adjuvanted vaccines in a consistent manner remains challenging. This chapter discusses methods to prepare aluminum salts and aluminum salt-adjuvanted vaccines, factors to consider during preparation, and methods to characterize the vaccines after preparation.

A method of lyophilizing vaccines containing aluminum salts into a dry powder without causing particle aggregation or decreasing the immunogenicity following reconstitution.

Many currently licensed and commercially available human vaccines contain aluminum salts as vaccine adjuvants. A major limitation with these vaccines is that they must not be exposed to freezing temperatures during transport or storage such that the liquid vaccine freezes, because freezing causes irreversible coagulation that damages the vaccines (e.g., loss of efficacy). Therefore, vaccines that contain aluminum salts as adjuvants are formulated as liquid suspensions and are required to be kept in cold chain (2-8°C) during transport and storage. Formulating vaccines adjuvanted with aluminum salts into dry powder that can be readily reconstituted before injection may address this limitation. Spray freeze-drying of vaccines with low concentrations of aluminum salts and high concentrations of trehalose alone, or a mixture of sugars and amino acids, as excipients can convert vaccines containing aluminum salts into dry powder, but fails to preserve the particle size and/or immunogenicity of the vaccines. In the present study, using ovalbumin as a model antigen adsorbed onto aluminum hydroxide or aluminum phosphate, a commercially available tetanus toxoid vaccine adjuvanted with potassium alum, a human hepatitis B vaccine adjuvanted with aluminum hydroxide, and a human papillomavirus vaccine adjuvanted with aluminum hydroxyphosphate sulfate, it was shown that vaccines containing a relatively high concentration of aluminum salts (i.e., up to ~1%, w/v, of aluminum hydroxide) can be converted into a dry powder by thin-film freezing followed by removal of the frozen solvent by lyophilization while using low levels of trehalose (i.e., as low as 2% w/v) as an excipient. Importantly, the thin-film freeze-drying process did not cause particle aggregation, nor decreased the immunogenicity of the vaccines. Moreover, repeated freezing-and-thawing of the dry vaccine powder did not cause aggregation. Thin-film freeze-drying is a viable platform technology to produce dry powders of vaccines that contain aluminum salts.

Skin penetration of topically applied white mustard extract and its effects on epidermal Langerhans cells and cytokines.

White mustard (Sinapis alba L.), a traditional Chinese medicine, is widely used in China for clinical prevention and treatment of the common winter diseases of asthma and bronchitis by percutaneous administration in the summer. The present study is to investigate the skin penetration behavior of white mustard extract to elucidate the possible mechanism underlying its immune regulation activity. The principle active compound of the extract, sinapine thiocyanate (ST), was used as a marker. The skin penetration of ST in white mustard extract was examined in vitro and in vivo. In vitro study on excised guinea pig hairless skin using Franz diffusion cell revealed ST can permeate through the skin and also accumulate in the skin. In vivo study was carried out on the guinea pig hairless skin for 24 h, and then skin was excised for frozen section, ST from the sections were extracted to quantify the amount of drug in different skin layers. The detailed distribution of ST showed that it accumulated in the epidermis, especially in the stratum corneum. After treatment with white mustard extract for 24h, the skin was stained with ATPase, and the morphometric parameters of epidermal LCs were compared to the untreated control through image-analysis system. A statistically significant reduction in LC density and increase in shape factor were observed. Cytokines related to LCs migration including interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) were also measured after white mustard extract treated at different time points. Compared to the untreated group, white mustard extract significantly enhanced the release of IL-1ß and TNFα. The morphometric changes of LCs and the local cytokine release after topical white mustard treatment may explain the activity of the white mustard extract against asthma and bronchitis.

Relationship between the size of nanoparticles and their adjuvant activity: data from a study with an improved experimental design.

There is a growing interest in identifying the relationship between the size of nanoparticles and their adjuvant activity, but the results from recent studies remain controversial. To address the controversy, it was thought that one should pay attention to the nanoparticle formulations to make sure that the antigen-loaded nanoparticles to be compared are not only different in particle size, but more importantly, as identical to each other as possible in all other formulation properties. In the present study, using ovalbumin (OVA) as a model antigen conjugated onto nanoparticles engineered from lecithin/glyceryl monostearate-in-water emulsions, we prepared OVA-nanoparticles of 230 nm and 708 nm. Before evaluating the immune responses induced by them in a mouse model, we made sure that: (i) the sizes of the two OVA-nanoparticles did not extensively overlap, (ii) the nanoparticles have similar zeta potentials and comparable antigen-loading, and (iii) the nanoparticles did not aggregate when suspended in simulated biological media. We then showed that when subcutaneously injected into mice, the 230 nm OVA-conjugated nanoparticles induced stronger OVA-specific antibody and cellular immune responses than the 708 nm OVA-nanoparticles. Future studies attempting to correlate the size of nanoparticles and their adjuvant activities need to consider formulation parameters to ensure that the particles are different only in size and are stable before and after injection.

Towards preserving the immunogenicity of protein antigens carried by nanoparticles while avoiding the cold chain.

Nanoparticles are an attractive vaccine carrier with potent adjuvant activity. Data from our previous studies showed that immunization of mice with lecithin/glyceryl monostearate-based nanoparticles with protein antigens conjugated onto their surface induced a strong, quick, and long-lasting antigen-specific immune response. In the present study, we evaluated the feasibility of preserving the immunogenicity of protein antigens carried by nanoparticles without refrigeration using these antigen-conjugated nanoparticles as a model. The nanoparticles were lyophilized, and the immunogenicity of the antigens was evaluated in a mouse model using bovine serum albumin or the Bacillus anthracis protective antigen protein as model antigens. With proper excipients, the nanoparticles can be lyophilized while maintaining the immunogenicity of the antigens. Moreover, the immunogenicity of the model antigen conjugated onto the nanoparticles was undamaged after a relatively extended period of storage at room temperature or under accelerated conditions (37 degrees C) when the nanoparticles were lyophilized with 5% mannitol plus 1% polyvinylpyrrolidone. To our knowledge, the present study represents an early attempt to preserve the immunogenicity of the protein antigens carried by nanoparticles without refrigeration.

Strong antibody responses induced by protein antigens conjugated onto the surface of lecithin-based nanoparticles.

An accumulation of research over the years has demonstrated the utility of nanoparticles as antigen carriers with adjuvant activity. Herein we defined the adjuvanticity of a novel lecithin-based nanoparticle engineered from emulsions. The nanoparticles were spheres of around 200nm. Model protein antigens, bovine serum albumin (BSA) or Bacillus anthracis protective antigen (PA) protein, were covalently conjugated onto the nanoparticles. Mice immunized with the BSA-conjugated nanoparticles developed strong anti-BSA antibody responses comparable to that induced by BSA adjuvanted with incomplete Freund's adjuvant and 6.5-fold stronger than that induced by BSA adsorbed onto aluminum hydroxide. Immunization of mice with the PA-conjugated nanoparticles elicited a quick, strong, and durable anti-PA antibody response that afforded protection of the mice against a lethal dose of anthrax lethal toxin challenge. The potent adjuvanticity of the nanoparticles was likely due to their ability to move the antigens into local draining lymph nodes, to enhance the uptake of the antigens by antigen-presenting cells (APCs), and to activate APCs. This novel nanoparticle system has the potential to serve as a universal protein-based vaccine carrier capable of inducing strong immune responses.

Nanoparticles engineered from lecithin-in-water emulsions as a potential delivery system for docetaxel.

Docetaxel is a potent anticancer drug. However, there continues to be a need for alternative docetaxel delivery systems to improve its efficacy. We reported the engineering of a novel spherical nanoparticle formulation ( approximately 270 nm) from lecithin-in-water emulsions. Docetaxel can be incorporated into the nanoparticles, and the resultant docetaxel-nanoparticles were stable when stored as an aqueous suspension. The release of the docetaxel from the nanoparticles was likely caused by a combination of diffusion and Case II transport. The docetaxel-in-nanoparticles were more effective in killing tumor cells in culture than free docetaxel. Moreover, the docetaxel-nanoparticles did not cause any significant red blood cell lysis or platelet aggregation in vitro, nor did they induce detectable acute liver damage when injected intravenously into mice. Finally, compared to free docetaxel, the intravenously injected docetaxel-nanoparticles increased the accumulation of the docetaxel in a model tumor in mice by 4.5-fold. These lecithin-based nanoparticles have the potential to be a novel biocompatible and efficacious delivery system for docetaxel.

Evaluation of the immune response induced by a nasal anthrax vaccine based on the protective antigen protein in anaesthetized and non-anaesthetized mice.

To better protect against inhalational anthrax infection, a nasal anthrax vaccine based on the protective antigen (PA) protein of Bacillus anthracis could be an attractive alternative to the current Anthrax-Vaccine-Adsorbed (AVA), which was licensed for cutaneous anthrax prevention. Previously, we have demonstrated that an anti-PA immune response comparable with that in mice subcutaneously immunized with PA protein adjuvanted with aluminium hydroxide was induced in both the systemic compartment and the mucosal secretions of the nose and lung of anaesthetized mice when they were nasally immunized with PA protein incorporated into previously reported LPD (Liposome-Protamine-DNA) particles. In this study, we evaluated the anti-PA immune response induced by the nasal PA/LPD particles in non-anaesthetized mice and compared it with that in anaesthetized mice. Our data showed that the anti-PA antibody response and the anthrax lethal toxin-neutralization activity induced by the nasal PA/LPD in non-anaesthetized mice was relatively weaker than that in anaesthetized mice. However, the splenocytes isolated from the nasally immunized mice, anaesthetized and non-anaesthetized, proliferated comparably after in-vitro re-stimulation. By evaluating the uptake of fluorescence-labelled LPD particles by phagocytes in the nasal and broncho-alveolar lavages of mice after the nasal administration, we concluded that the relatively weaker anti-PA immune response in the non-anaesthetized mice might be partially attributed to the reduced retention of the PA/LPD particles in the nasal cavity of the non-anaesthetized mice. Data collected in this study are expected to be useful for future anthrax nasal vaccine studies when mice are used as a model.

Novel D-penicillamine carrying nanoparticles for metal chelation therapy in Alzheimer's and other CNS diseases.

Metal ions accumulate in the brain with aging and in several neurodegenerative diseases. Aside from the copper storage disease, Wilson's disease, recent attention has focused on the accumulation of zinc, copper and iron in the Alzheimer's disease (AD) brain and the accumulation of iron in Parkinson's disease. In particular, the parenchymal deposition of beta-amyloid (Abeta) and its interaction with metal ions has been postulated to play a role in the progression of AD. Thus, the strategy of lowering brain metal ions and targeting the interaction of Abeta peptide and metal ions through the administration of chelators has merit. Our recent finding that nanoparticle delivery systems can cross the blood-brain barrier has led us to investigate whether chelators delivered conjugated to nanoparticles could act to reverse metal ion induced protein precipitation. In the present studies, the Cu (I) chelator D-penicillamine was covalently conjugated to nanoparticles via a disulfide bond or a thioether bond. Nanoparticle-chelator conjugates were stable between pH 6-8 in aqueous suspension if stored at 4 degrees C, and did not aggregate when challenged with salts and serum. Release of D-penicillamine from the nanoparticles was achieved using reducing agents such as dithiothreitol (as a model for glutathione). Nanoparticles treated only under reducing conditions that released the conjugated D-penicillamine were able to effectively resolubilize copper-Abeta (1-42) aggregates. These results indicate that nanoparticles have potential to deliver D-penicillamine to the brain for the prevention of Abeta (1-42) accumulation, as well as to reduce metal ion accumulation in other CNS diseases.