Effect of Cholesterol Content of Lipid Composition in mRNA-LNPs on the Protein Expression in the Injected Site and Liver After Local Administration in Mice.

Delivery of messenger RNA (mRNA) using lipid nanoparticles (LNPs) is expected to be applied to various diseases following the successful clinical use of the mRNA COVID-19 vaccines. This study aimed to evaluate the effect of the cholesterol molar percentage of mRNA-LNPs on protein expression in hepatocellular carcinoma-derived cells and in the liver after intramuscular or subcutaneous administration of mRNA-LNPs in mice. For mRNA-LNPs with cholesterol molar percentages reduced to 10 mol% and 20 mol%, we formulated neutral charge particles with a diameter of approximately 100 nm and polydispersity index (PDI) <0.25. After the intramuscular or subcutaneous administration of mRNA-LNPs with different cholesterol molar percentages in mice, protein expression in the liver decreased as the cholesterol molar percentage in mRNA-LNPs decreased from 40 mol% to 20 mol% and 10 mol%, suggesting that reducing the cholesterol molar percentage in mRNA-LNPs decreases protein expression in the liver. Furthermore, in HepG2 cells, protein expression decreased as cholesterol in mRNA-LNPs was reduced by 40 mol%, 20 mol%, and 10 mol%. These results suggest that the downregulated expression of mRNA-LNPs with low cholesterol content in the liver involves degradation in systemic circulating blood and decreased protein expression after hepatocyte distribution.

Desenvolvimento e caracterização de lipossomas peguilados para veiculação de L-asparaginase / Development and characterization of peg-grafted liposomes for lasparaginase encapsulation

A Leucemia Linfoide Aguda (LLA) é um câncer de maior incidência em crianças, e tem a Lasparaginase (ASNase) como fármaco amplamente utilizado no tratamento dos afetados. A ASNase catalisa a hidrólise do aminoácido L-asparagina (Asn), presente na corrente sanguínea, a ausência do aminoácido no meio extracelular leva à morte células leucêmicas, que necessitam deste aminoácido para as funções celulares. Fatores envolvendo a eficiência do tratamento com ASNase como reações adversas e curta meia-vida, principalmente devido ao reconhecimento pelo sistema imune e degradação por proteases, limitam a sua eficácia. A encapsulação da enzima em lipossomas pode conferir proteção à degradação, melhorar seu perfil farmacocinético e diminuir os efeitos adversos, de forma a melhorar o tratamento da LLA sendo este o objetivo desse trabalho. Lipossomas de DOPC (1,2-dioleoil-sn-glicero-3-fosfocolina) e DMPC (1,2-dimiristoil-snglicero-3-fosfocolina) foram desenvolvidos empregando-se o método de hidratação do filme lipídico e diferentes protocolos de preparo contendo ou não diferentes concentrações de 18:0 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polietilenogicol)-2000] (DSPE-PEG). Os lipossomas produzidos foram utilizados para encapsular a ASNase e os sistemas contendo ou não ASNase encapsulada foram caracterizados por espalhamento de luz dinâmico (DLS), potencial zeta, microscopia eletrônica de transmissão (MET) e criomicroscopia de transmissão. Adicionalmente, foram avaliados a taxa de encapsulação e o perfil de permeabilidade das vesículas à L-asparagina. As análises de DLS mostraram que as nanoestruturas formadas empregando-se agitação magnética a partir de sistemas contendo 10% e 20% de DSPE-PEG possuem diâmetro hidrodinâmico menor (~ 25 nm a 60 nm) que os mesmos sistemas sem o fosfolipídio peguilado (~190 nm a 222 nm), demonstrando a relação entre a diminuição do tamanho e o aumento da quantidade de fosfolipídio peguilado e possível formação de estruturas micelares ou bicelares. O emprego de agitação em vórtex para hidratação do filme lipídico, adição do antioxidante -tocoferol e redução da concentração de DSPE-PEG (5% e 10%) levou à formação de sistemas com diâmetro hidrodinâmico maior, sendo esse protocolo e concentrações de PEG definidos como padrão. As análises de MET comprovaram a formação de lipossomas com diâmetro hidrodinâmico semelhante ao observado por DLS; com a utilização da criomicroscopia foi possível observar os lipossomas sem deformações. Os lipossomas de DMPC/DSPE-PEG 10% apresentaram maior permeabilidade à L-asparagina ao longo do tempo e, portanto, poderiam funcionar como nanoreatores, depletando o aminoácido da circulação. Estudos in vitro com células tumorais devem ser realizados e em seguida estudos in vivo, para confirmar este potencial

L-asparaginase (ASNase) is a first-choice drug, combined with other drugs, in therapeutic schemes to treat Acute Lymphoblastic Leukemia (ALL) in children and adolescents. ASNase catalyzes the hydrolysis of L-asparagine (Asn) in the bloodstream; since ALL cells cannot synthesize this amino acid, protein synthesis is impaired leading to leukemic cells death by apoptosis. In spite of its therapeutic importance, treatment with ASNase is associated to side effects, mainly hypersensitivity and immunogenicity. Another drawback refers to degradation by plasma proteases that altogether with immunogenicity shortens the enzyme half-life. Encapsulation of ASNase in liposomes, vesicular nanostructures formed by the self-aggregation of phospholipids, is an attractive alternative that possibly will protect the enzyme from plasma proteases, resulting on better pharmacokinetics profile. In this work, we prepared by thin film hydration liposomal formulations of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC) containing or not different concentrations of 18:0 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), and encapsulated ASNase by electroporation. The systems containing or not ASNase were analyzed by Dynamic Light Scattering, zeta potential and Electron Microscopy. The encapsulation efficiency and vesicles permeability were also evaluated. According to the DLS analysis, the nanostructures formed by film hydration under magnetic stirring employing 10% or 20% DSPE-PEG presented smaller hydrodynamic diameter (~ 25 nm to 60 nm) than the same systems without the pegylated phospholipid (~ 190 nm to 222 nm), demonstrating the relation between size and the amount of pegylated phospholipid that results in formation of micellar or bicellar structures. The protocol was stabilize by hydration of the lipid film under vortex agitation, addition of the antioxidant - tocopherol and reduction of the concentration of DSPE-PEG (5% and 10%), what altogether led to the formation of nanostructures of higher hydrodynamic diameter and monodisperse systems. TEM analyzes confirmed the formation of liposomes with hydrodynamic diameter similar to that observed by DLS; with the use of cryomicroscopy it was possible to observe the liposomes without deformations. Liposomes of DMPC/DSPE-PEG 10% showed permeability to L-asparagine over time and, therefore, could function as nanoreactors, depleting the circulating amino acid

Identification of Abies sibirica L. Polyprenols and Characterisation of Polyprenol-Containing Liposomes.

The needles of conifer trees are one of the richest sources of natural polyprenols. Polyprenol homologs from Abies sibirica L. lipophilic 80% purified extract were analyzed and quantified. In total, 10 peaks (Prenol-11 to Prenol-20) were observed in the ultra-high-performance liquid chromatography-diode array detector (UHPLC-DAD) chromatogram of Siberian fir with the most abundant compound being Prenol-15 (relative amount 37.23 + 0.56% of the total polyprenol yield). Abies sibirica L. polyprenol solubility and incorporation efficiency into liposomes were studied in various commercially available lecithin mixtures (Phosal IP40, Phosal 75SA, and Lipoid P45). The resulting multilamellar polyprenol liposomes were morphologically characterized by Light and Transmission Electron Microscopy, and the liposome size was discovered to be polymodal with the main peak at 1360 nm (90% of the volume). As polyprenols are fully soluble only in lipids, a liposomal formulation based upon co-solubilization and a modified ethanol injection method of polyprenols into the ethanol-phospholipid system was developed for the entrapment and delivery of polyprenols for potential commercial applications in food supplement and cosmetic industries.

Preparation and characterization of liposomes loaded with silver nanoparticles obtained by green synthesis

The objective of this work was to develop and characterize liposomes loaded with silver nanoparticles (LAgNPs) to show improvement in stability characteristics. AgNPs were prepared by the green synthesis method with Aloe vera gel extract and exposure to sunlight. Liposomes were prepared by the modified reverse phase method. Particle size, polydispersity index, zeta potential, as well as the scanning electron microscopy (SEM) morphological aspects of AgNPs and LAgNPs were evaluated. In addition, was used flame atomic absorption spectroscopy to determine the amount of AgNP that was encapsulated in liposomes. The AgNPs presented as amorphous and polydisperse structures, with a mean diameter of 278.46 nm and zeta potential of -18.3 mV. LAgNPs had a mean diameter between 321 and 373 nm, the polydispersity index close to 0.2 and a zeta potential around -40 mV, which indicates greater stability to the AgNPs. The images obtained by SEM show semicircular structures for AgNPs and well-defined spherical shape for LAgNPs. The percentage of encapsulation was between 51.81 to 58.83%. These results showed that LAgNPs were obtained with adequate physicochemical characteristics as a release system.

Preparation and evaluation of 2-methoxyestradiol-loaded pH-sensitive liposomes

The development and clinical application of 2-methoxyestradiol (2-ME) as a new type of antitumor drug are limited due to its poor solubility, rapid metabolism in vivo, and large oral dosage. 2-ME-loaded pH-sensitive liposomes (2-ME-PSLs) was prepared containing the lipids, Lipoid E-80 (E-80), cholesteryl hemisuccinate (CHEMS), and cholesterol (CHOL) via thin-film ultrasonic dispersion. First, preparation conditions of 2-ME-PSLs were optimized by orthogonal test. Then 2-ME-PSL was characterized, and the release behavior and stability of 2-ME-PSL in vitro were evaluated. The optimal preparation conditions for 2-ME-PSLs were as follows: 2-ME : E-80+CHEMS 1:15; CHOL : E-80+CHEMS 1:5; ultrasonication time 20 minutes. The mean particle size, PDI, zeta potential, and entrapment efficiency (EE) of 2-ME-PSLs were 116 ± 9 nm, 0.161 ± 0.025, −22.4 ± 1.7 mV, and 98.6 ± 0.5%, respectively. As viewed under a transmission electron microscope, 2-ME-PSLs were well dispersed and almost spherical. They exhibited significant pH-sensitive properties and were fairly stable when diluted with a physiological solution. In conclusion, 2-ME-PSLs were successfully prepared and possessed a favorable pH sensitivity and good dissolution stability with a normal solution

Directing Nanoparticle Biodistribution through Evasion and Exploitation of Stab2-Dependent Nanoparticle Uptake.

Up to 99% of systemically administered nanoparticles are cleared through the liver. Within the liver, most nanoparticles are thought to be sequestered by macrophages (Kupffer cells), although significant nanoparticle interactions with other hepatic cells have also been observed. To achieve effective cell-specific targeting of drugs through nanoparticle encapsulation, improved mechanistic understanding of nanoparticle-liver interactions is required. Here, we show the caudal vein of the embryonic zebrafish ( Danio rerio) can be used as a model for assessing nanoparticle interactions with mammalian liver sinusoidal (or scavenger) endothelial cells (SECs) and macrophages. We observe that anionic nanoparticles are primarily taken up by SECs and identify an essential requirement for the scavenger receptor, stabilin-2 ( stab2) in this process. Importantly, nanoparticle-SEC interactions can be blocked by dextran sulfate, a competitive inhibitor of stab2 and other scavenger receptors. Finally, we exploit nanoparticle-SEC interactions to demonstrate targeted intracellular drug delivery resulting in the selective deletion of a single blood vessel in the zebrafish embryo. Together, we propose stab2 inhibition or targeting as a general approach for modifying nanoparticle-liver interactions of a wide range of nanomedicines.

Intrabilayer 64Cu Labeling of Photoactivatable, Doxorubicin-Loaded Stealth Liposomes.

Doxorubicin (Dox)-loaded stealth liposomes (similar to those in clinical use) can incorporate small amounts of porphyrin-phospholipid (PoP) to enable chemophototherapy (CPT). PoP is also an intrinsic and intrabilayer 64Cu chelator, although how radiolabeling impacts drug delivery has not yet been assessed. Here, we show that 64Cu can radiolabel the stable bilayer of preformed Dox-loaded PoP liposomes with inclusion of 1% ethanol without inducing drug leakage. Dox-PoP liposomes labeled with intrabilayer copper behaved nearly identically to unlabeled ones in vitro and in vivo with respect to physical parameters, pharmacokinetics, and CPT efficacy. Positron emission tomography and near-infrared fluorescence imaging visualized orthotopic mammary tumors in mice with passive liposome accumulation following administration. A single CPT treatment with 665 nm light (200 J/cm2) strongly inhibited primary tumor growth. Liposomes accumulated in lung metastases, based on NIR imaging. These results establish the feasibility of CPT interventions guided by intrinsic multimodal imaging of Dox-loaded stealth PoP liposomes.

A lyophilized sterically stabilized liposome-containing docetaxel: in vitro and in vivo evaluation.

OBJECTIVES: In this study, an improved lyophilized PEGylated liposomal formulation of docetaxel (DOC) has been developed. METHODS: PEGylated docetaxel liposome (PL-DOC) was prepared by thin-film evaporation method and lyophilization. The effect of various components of the lipids and their compatibility with DOC on the entrapment efficiency (EE) of liposome was investigated. The lyophilized PL-DOC was characterized by morphology, particle size, zeta potential, EE, release in vitro and stability. Pharmacokinetics and biodistribution in vivo of lyophilized PL-DOC were also investigated. RESULTS: The optimal liposome formulation was egg phosphatidylcholine (EPC):cholesterol (CH):DSPE-PEG2000:DOC = 56:40:4:4 (molar ratio). Sucrose and mannitol were chosen as cryoprotectant in the lyophilization (cryoprotectant-to-lipid (C/L) mass ratio = 8:1). The size of lyophilized PL-DOC was 152.3 ± 1.0 nm with negative charge and the EE was 89.75 ± 1.79%. Compared with nonlyophilized PL-DOC, the lyophilized PL-DOC was more stable at 4 °C for six months. The lyophilized PL-DOC also showed the good stability after reconstituted by 5% glucose injection. In vitro release study of PL-DOC showed that PL-DOC had a sustained release effect. After i.v. administration at the dose of 10 mg/kg in rats, a significant increase in the AUC0-∞, MRT0-∞ and t1/2 was observed in PL-DOC group compared with conventional docetaxel liposome (CL-DOC) and DOC injection (DOC-I) group. Biodistribution studies in mice showed that PL-DOC significantly decreased the uptake by the organs of mononuclear phagocytic system (MPS), such as liver and spleen, while prolonging the retention time of DOC in the plasma. CONCLUSION: Our PEGylated liposome formulation reported in this study could potentially produce viable clinical strategies for improved delivery of DOC for the treatment of human cancer.

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake.

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations.

Nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) of liposomes: applicability of the technique for nano vesicle batch control.

Liposomes are biodegradable nanoparticle vesicles consisting of a lipid bilayer encapsulating an aqueous core. Entrapped cargo material is shielded from the extra-vesicular medium and sustained release of encapsulated material can be achieved. However, application of liposomes as nano-carriers demands their characterization concerning size and size distribution, particle-number concentration, occurrence of vesicle building blocks in solution and determination of the resulting vesicle encapsulation capacity. These questions can be targeted via gas-phase electrophoretic mobility molecular analysis (GEMMA) based on a nano electrospray (nES) charge-reduction source. This instrument separates single-charged nanoparticles in the gas-phase according to size in a high-laminar sheath-flow by means of an orthogonal, tunable electric field. nES GEMMA analysis enables to confirm liposome integrity after passage through the instrument (in combination with atomic force microscopy) as well as to exclude vesicle aggregation. Additionally, nanoparticle diameters at peak apexes and size distribution data are obtained. Differences of hydrodynamic and dry particle diameter values, as well as the effect of number- and mass-based concentration data analysis on obtained liposome diameters are shown. Furthermore, the repeatability of liposome preparation is studied, especially upon incorporation of PEGylated lipids in the bilayer. Finally, the instruments applicability to monitor mechanical stress applied to vesicles is demonstrated.

Capillary electrophoresis method to determine siRNA complexation with cationic liposomes.

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally nanoparticles. The nanoparticulate complex requires an optimal physiochemical characterization and the complexation efficiency has to be precisely determined. The methods usually used to measure complexation in gel electrophoresis and RiboGreen® fluorescence-based assay. However, those approaches are not automated and present some drawbacks such as the low throughput and the use of carcinogenic reagents. The aim of this study is to develop a new simple and fast method to accurately quantify the complexation efficiency. In this study, capillary electrophoresis (CE) was used to determine the siRNA complexation with cationic liposomes. The short-end injection mode applied enabled siRNA detection in less than 5 min. Moreover, the CE technique offers many advantages compared with the other classical methods. It is automated, does not require sample preparation and expensive reagents. Moreover, no mutagenic risk is associated with the CE approach since no carcinogenic product is used. Finally, this methodology can also be extended for the characterization of other types of nanoparticles encapsulating siRNA, such as cationic polymeric nanoparticles.

Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics.

Nano-sized extracelullar vesicles (EVs) released by various cell types play important roles in a plethora of (patho)physiological processes and are increasingly recognized as biomarkers for disease. In addition, engineered EV and EV-inspired liposomes hold great potential as drug delivery systems. Major technologies developed for high-throughput analysis of individual EV include nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (tRPS) and high-resolution flow cytometry (hFC). Currently, there is a need for comparative studies on the available technologies to improve standardization of vesicle analysis in diagnostic or therapeutic settings. We investigated the possibilities, limitations and comparability of NTA, tRPS and hFC for analysis of tumor cell-derived EVs and synthetic mimics (i.e. differently sized liposomes). NTA and tRPS instrument settings were identified that significantly affected the quantification of these particles. Furthermore, we detailed the differences in absolute quantification of EVs and liposomes using the three technologies. This study increases our understanding of possibilities and pitfalls of NTA, tRPS and hFC, which will benefit standardized and large-scale clinical application of (engineered) EVs and EV-mimics in the future.

Formulation design and characterization of a non-ionic surfactant based vesicular system for the sustained delivery of a new chondroprotective agent

Diacerein is used for symptomatic relief and cartilage regeneration in osteoarthritis. Due to gastrointestinal side effects, poor aqueous solubility and low bioavailability, its clinical usage has been restricted. The objective of the present study was to enhance its dissolution profile and to attain sustained release by designing a novel delivery system based on niosomes. Five niosomal formulations (F1-F5) with non-ionic surfactant (sorbitan monostearate) and cholesterol in varying ratios of 5:5, 6:4, 7:3, 8:2 and 9:1 were developed by the reverse-phase evaporation technique. The size and polydispersivity index (PDI) were found in the range of 0.608 µm to 1.010 µm and 0.409 to 0.781, respectively. Scanning electron microscopy (SEM) of the selected formulation (F3) revealed spherical vesicles, and 79.8% entrapment was achieved with F3 (7:3). Dissolution studies using the dialysis method showed sustained release behaviour for all formulations. The optimized surfactant-to-cholesterol concentration (7:3) in formulation F3sustained the drug-release time (T50%) up to 10 hours. Kinetic modelling exhibited a zero-order release (R2=0.9834) and the release exponent 'n' of the Korsmayer-Peppas model (n=0.90) confirmed non-fickian and anomalous release. The results of this study suggest that diacerein can be successfully entrapped into niosomes using sorbitan monostearate and that these niosomes have the potential to deliver diacerein efficiently at the absorption site.

A diacereína é usada para o alívio sintomático e para a regeneração da cartilagem na osteoartrite. Devido aos efeitos adversos gastrointestinais, baixa solubilidade aquosa e biodisponibilidade, o seu uso clínico tem sido restrito. O objetivo do presente estudo foi melhorar o perfil de dissolução deste fármaco e obter liberação prolongada através do planejamento de um novo sistema de liberação designado de niossoma. Cinco formulações distintas de niossomas (F1 a F5) contendo tensoativos não iônicos (monoestearato de sorbitano) e colesterol, em diferentes proporções, de 5:5, 6:4, 7:3, 8:2 e 9:1, foram desenvolvidas através da técnica de evaporacão de fase reversa. Os tamanhos e índices de polidispersibilidade (PDI) obtidos variam entre 0,608 e 1,01 µm e entre 0,409 e 0,7781, respectivamente. Imagens de microscopia electrônica de varrimento (SEM) da formulação selecionada (F3) revelaram vesículas esféricas. Obteve-se encapsulação de 79,8% com a formulação F3 (7:3). Estudos de dissolução usando o método de diálise demonstraram padrão de liberacão prolongada para todas as formulações. A proporção de tensoativo e colesterol (7:3) na formulacão F3 prolongou o tempo de liberação do fármaco (T50%) até 10 horas. Estudos de modelação cinética demonstraram ordem de liberacão zero (R2=0,9834) e o expoente de liberação "n" do modelo de Korsmayer-Peppas (n=0.90) confirmou a liberação não-fickiana e anômala. Os resultados deste estudo sugerem que a diacereína pode ser encapsulada com sucesso no interior de niossomas, utilizando monostearato de sorbitano, o qual tem potencial para liberar, eficientemente, a diacereína no local de absorção.

Development and validation of an analytical method for quantification of arsenic and antimony in liposomes using inductively coupled plasma-optical emission spectrometry.

Arsenic and antimony compounds are used to treat endemic diseases, such as cancer, leishmaniasis, and schistosomiasis, in spite of their toxicity. Several studies seeking the development and characterization of nanocarrier systems such as liposomes are being carried out with the aim of developing new drug delivery systems and minimizing the toxicity of these drugs. However, the lack of reference methods to quantify these semimetals within a liposomal matrix hinders the QC of these formulations. Therefore, the validation of an analytical method for arsenic and antimony quantification in liposomal matrix by inductively coupled plasma-optical emission spectrometry is presented here. The linearity, specificity, LOD, LOQ, accuracy, and precision were determined according to the International Conference on Harmonization norms and the Brazilian Health Surveillance Agency (Resolution 899). The LOD values were 0.02 and 0.06 mg/L for antimony and arsenic, respectively. The LOQ for both was 3.0 mg/L, with an adequate accuracy within 98.26 and 101.32% for different levels of antimony and 99.98 and 100.36% for arsenic. Precision (CV) was lower than 5.0%. The developed and validated method was shown to be reproducible for quantification of arsenic and antimony in liposome pharmaceutical dosage forms.

Complement activation-related pseudoallergy in dogs following intravenous administration of a liposomal formulation of meglumine antimoniate / Pseudoalergia relacionada à ativação do complemento em cães após administração intravenosa de uma formulação lipossomal de antimoniato de meglumina

The increasing use of nanotechnologies in advanced therapies has allowed the observation of specific adverse reactions related to nanostructures. The toxicity of a novel liposome formulation of meglumine antimoniate in dogs with visceral leishmaniasis after single dose has been investigated. Groups of 12 animals received by the intravenous route a single dose of liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg), empty liposomes (GII) or isotonic saline (GIII). Evaluation of hematological and biochemical parameters showed no significant changes 4 days after administration. No undesired effects were registered in the GIII. However, adverse reactions were observed in 67.7% of dogs from both groups that received liposomal formulations. The side effects began moments after bolus administration and disappeared during the first 15 minutes after treatment. Prostation, sialorrhea and defecation were the most frequent clinical signs, registered in 33.3% and 41.6 % of animals from the groups GI and GII, respectively. Tachypnea, mydriasis, miosis, vomiting and cyanosis were also registered in both groups. The adverse reactions observed in this study were attributed to the activation of the complement system by lipid vesicles in a phenomenon known as Complement Activation-Related Pseudoallergy (CARPA). The influence of the physical-chemical characteristics of liposomal formulation in the triggering of CARPA is discussed.

O crescente uso das nanotecnologias nas terapias avançadas tem permitido a observação de reações adversas específicas relacionadas às nanoestruturas. A toxicidade de uma nova formulação lipossomal de antimoniato de meglumina após dose única foi avaliada em cães com leishmaniose visceral. Grupos de 12 animais receberam por via intravenosa uma dose única de antimoniato de meglumina lipossomal (grupo I [GI], 6,5 mg Sb/kg), lipossomas vazios (GII) ou solução salina isotônica (GIII). A avaliação de parâmetros hematológicos e bioquímicos não revelou alterações significativas quatro dias após a administração. Nenhum efeito indesejável foi registrado no GIII. No entanto, reações adversas foram observadas em 67,7% dos cães de ambos os grupos que receberam formulações lipossomais. Os efeitos colaterais iniciaram momentos após a administração em "bolus" e desapareceram no decurso dos primeiros 15 minutos após o tratamento. Prostração, sialorréia e defecação foram os sinais clínicos mais frequentes, registrados em 33,3% e 41,6% dos animais dos grupos GI e GII, respectivamente. Taquipnéia, midríase, miose, vômitos e cianose também foram registrados em ambos os grupos. As reações adversas observadas neste trabalho foram atribuídas à ativação do sistema complemento pelas vesículas lipídicas em fenômeno conhecido como Pseudoalergia Relacionada à Ativação do Complemento (PARAC). A influência das características físico-químicas da formulação lipossomal no desencadeamento de PARAC é abordada.

Convection-enhanced delivery of targeted quantum dot-immunoliposome hybrid nanoparticles to intracranial brain tumor models.

AIM: The aim of this work is to evaluate combining targeting strategy and convection-enhanced delivery in brain tumor models by imaging quantum dot-immunoliposome hybrid nanoparticles. MATERIALS & METHODS: An EGF receptor-targeted, quantum dot-immunoliposome hybrid nanoparticle (QD-IL) was synthesized. In vitro uptake was measured by flow cytometry and intracellular localization was imaged by confocal microscopy. In the in vivo study, QD-ILs were delivered to intracranial xenografts via convection-enhanced delivery and fluorescence was monitored noninvasively in real-time. RESULTS: QD-ILs exhibited specific and efficient uptake in vitro and exhibited approximately 1.3- to 5.0-fold higher total fluorescence compared with nontargeted counterpart in intracranial brain tumor xenografts in vivo. CONCLUSION: QD-ILs serve as an effective imaging agent in vitro and in vivo, and the data suggest that ligand-directed liposomal nanoparticles in conjunction with convection-enhanced delivery may offer therapeutic benefits for glioblastoma treatment as a result of specific and efficient uptake by malignant cells.

Simultaneous quantification of tumor uptake for targeted and nontargeted liposomes and their encapsulated contents by ICPMS.

Liposomes are intensively being developed for biomedical applications including drug and gene delivery. However, targeted liposomal delivery in cancer treatment is a very complicated multistep process. Unfavorable liposome biodistribution upon intravenous administration and membrane destabilization in blood circulation could result in only a very small fraction of cargo reaching the tumors. It would therefore be desirable to develop new quantitative strategies to track liposomal delivery systems to improve the therapeutic index and decrease systemic toxicity. Here, we developed a simple and nonradiative method to quantify the tumor uptake of targeted and nontargeted control liposomes as well as their encapsulated contents simultaneously. Specifically, four different chelated lanthanide metals were encapsulated or surface-conjugated onto tumor-targeted and nontargeted liposomes, respectively. The two liposome formulations were then injected into tumor-bearing mice simultaneously, and their tumor delivery was determined quantitatively via inductively coupled plasma mass spectroscopy (ICPMS), allowing for direct comparisons. Tumor uptake of the liposomes themselves and their encapsulated contents was consistent with targeted and nontargeted liposome formulations that were injected individually.

Characterization of polymerized liposomes using a combination of dc and cyclical electrical field-flow fractionation.

Characterization of polymerized liposomes (PolyPIPosomes) was carried out using a combination of normal dc electrical field-flow fractionation and cyclical electrical field-flow fractionation (CyElFFF) as an analytical technique. The constant nature of the carrier fluid and channel configuration for this technique eliminates many variables associated with multidimensional analysis. CyElFFF uses an oscillating field to induce separation and is performed in the same channel as standard dc electrical field-flow fractionation separation. Theory and experimental methods to characterize nanoparticles in terms of their sizes and electrophoretic mobilities are discussed in this paper. Polystyrene nanoparticles are used for system calibration and characterization of the separation performance, whereas polymerized liposomes are used to demonstrate the applicability of the system to biomedical samples. This paper is also the first to report separation and a higher effective field when CyElFFF is operated at very low applied voltages. The technique is shown to have the ability to quantify both particle size and electrophoretic mobility distributions for colloidal polystyrene nanoparticles and PolyPIPosomes.

Amyloid detection using a Peltier-based device.

Amyloid aggregation of polypeptides is related to a growing number of pathologic states known as amyloid disorders. At present, it is clear that any proteins submitted to appropriate physicochemical environment can acquire fibrilar conformation. Fourier transform infrared spectroscopy (FTIR) has been a widely used technique to study temperature- induced amyloid-fibrils formation in vitro. In this way, strict changes and temperature controls are required to characterize the physicochemical basis of the amyloid-fibrils formation. In this article, the development of a highly efficient and accurate Peltier-based system to improve FTIR measurements is presented (see An Old Physics Phenomenon Applied to a Serious Biomedical Pathology. The accuracy of the thermostatic control was tested with biophysical parameters on biological samples probing its reproducibility. The design of the present device contributes to maintain the FTIR environment stable, which represents a real contribution to improve the spectral quality and thus, the reliability of the results.

The utility of an isolated mitochondrial fraction in the preparation of liposomes for the specific delivery of bioactives to mitochondria in live mammalian cells.

PURPOSE: To develop and evaluate liposomal formulations prepared with an isolated mitochondrial fraction as a mitochondria-specific delivery vehicle. METHODS: Liposomes were prepared with either a crude mitochondrial fraction extracted from cells or lipids extracted from the crude mitochondrial fraction and were then characterized by determining their size and zeta potential. The cell uptake of the liposomes and loaded bioactive was studied using flow cytometry and confocal microscopy. The cytotoxicity of the formulations was tested by MTS cytotoxicity assay. RESULTS: Liposomes prepared with the mitochondrial extracts were non-toxic and colocalized with mitochondria in F98 cells. Addition of DOTAP to the liposomes facilitated DNA complexation and the DNA delivered intracellularly co-localized with mitochondria. CONCLUSION: The results from this study establish the potential of using a mitochondrial fraction isolated from cells to prepare liposomes capable of delivering biologically active molecules to mitochondria of live mammalian cells.