Development and estimation of nanosomal rifampicin]

A lab-scale method for preparation of rifampicin-loaded polybutylcyanoacrylate nanoparticles (nanosames) was developed. The biodistribution of the nanosome-entrapped rifampicin after its intravenous administration was studied on healthy mice. The nanoparticles provided significant liver and spleen accumulation of rifampicin. Modification of the nanoparticles surface with poloxamer 407 or poloxamine 908 led to optimization of the biodistribution: the concentrations of rifampicin in the lungs and plasma increased, whereas the liver accumulation decreased vs. the unmodified nanoparticles. The increased lung accumulation of rifampicin enhanced bacterial clearance in the lungs of the mice infected with M. tuberculosis. The results showed that the use of the nanoparticles for optimization of the drug biodistribution was effective for increasing the efficacy of antiinfective chemotherapy.     

Pharmacokinetics of kanamycin during targeted delivery to the liver in erythrocyte ghosts in animals with experimental acute cholecystitis

Pharmacokinetics of kanamycin was studied after its targeted delivery to the liver in autological erythrocyte ghosts on 25 noninbred dogs with experimental acute cholecystitis in comparison to the routine intravenous administration of the antibiotic in solution. Kanamycin concentrations in the tissues of the liver, pancreas, spleen, kidneys and lungs as well as in bile and blood serum were determined by the agar diffusion method 24, 48 and 72 hours after the last administration. It was found that the targeted delivery of kanamycin in blood shadows made it possible to provide high concentrations of the antibiotic for prolonged periods in the liver and biliary ducts and to more efficiently arrest the clinical manifestations of acute cholecystitis as well as normalize the laboratory indices. The data showed that using blood shadows as a reliable system for targeted delivery of antibiotics to the liver was advisable in purulent inflammatory affections of the biliary ducts.     

Biodistribution and Clearance of TiO2 Nanoparticles in Rats after Intravenous Injection

Titanium dioxide (TiO₂) nanoparticles are used in many applications. Due to their small size, easy body penetration and toxicological adverse effects have been suspected. Numerous studies have tried to characterize TiO₂ translocation after oral, dermal or respiratory exposure. In this study, we focused on TiO₂ nanoparticle biodistribution, clearance and toxicological effects after intravenous injection, considering TiO₂ translocation in the blood occurs. Using ICP-OES, transmission electron microscopy, and histological methods, we found TiO₂ accumulation in liver, lungs and spleen. We estimated TiO₂ nanoparticles’ half life in the body to about 10 days. Clinical biomarkers were also quantified for 56 days to identify potential toxicological impact on lungs, blood, liver, spleen and kidneys. Results showed absence of toxicological effects after TiO₂ intravenous injection at concentrations of 7.7 to 9.4 mg/kg.     

纳米氧化锌经口染毒对C57BL/6J小鼠外周脏器的影响*

目的:探讨纳米氧化锌经口染毒60 d对C57BL/6J小鼠多种外周脏器的损伤作用。方法:20只雄性C57BL/6J小鼠随机分为对照组和实验组,每组10只,实验组将纳米氧化锌溶液以20 mg/kg体重的剂量连续灌胃染毒60 d,对照组给予相应量的生理盐水;小鼠每周称重一次,染毒结束后,眼球取血,检测血糖、血脂、肝功能和肾功能相关指标,以及血清中炎症因子PAF、IL-6和TNF-α含量;取心脏、肝脏、脾脏、肺、肾脏和小肠组织制备病理切片,HE染色后,观察组织形态学变化。结果:实验组和对照组之间的体重无显著性差异;与正常对照组比较,实验组大鼠血中白蛋白(ALB)、白蛋白/球蛋白比值(A/G)、碱性磷酸酶(ALP)、谷草/谷丙转氨酶比值(S/L)、尿酸(UA)和尿素氮(BUN)含量明显升高(P＜0.05或 P＜0.01);两组间血清中炎症因子含量无显著差异。病理学检查发现,实验组心肌中部分区域出现浊肿,肝脏出现轻度炎性病变(灶性或小灶性坏死),脾脏色素沉着减少,肺部出现轻或中度间质性炎症,肾脏和小肠未见明显病理改变。结论:纳米氧化锌经口染毒60 d未引起C57BL/6J小鼠血液系统炎症,但可诱导心脏、肝脏、脾脏和肺脏出现轻度的病理变化,并导致肝脏和肾脏的功能异常。     

自组装海藻酸钠纳米粒在正常小鼠体内分布的研究

目的:研究自组装海藻酸纳米粒(sSAN)在小鼠的体内分布情况,探讨sSAN作为维生素D3药物载体的可行性。方法:用异硫氰基荧光素(FITC)标记sSAN和负载维生素D3的海藻酸纳米粒(sSAN-VD3),将两种标记好的纳米粒分别给予小鼠灌胃,在不同的时间将小鼠处死,分别取血清和肝、肺、肾、脾,各脏器经匀浆后,用荧光分光光度计测定其荧光强度,计算血清和各组织中sSAN和sSAN-VD3的含量。结果:经灌胃给药后在小鼠血清、肝、肾、肺中均检测到上述药物,而脾中没有检测到。给药后0.5h和1h,sSAN-VD3-FITC及sSAN-FITC在肝、肺和血清中的含量持续增加,以1h时达峰值浓度,给药2h、4h后,两种制剂的浓度逐渐降低。在肾脏中的含量随着时间的延长逐渐增加,于2h时达峰值浓度,随后逐渐降低。结论:sSAN及sSAN-VD3经小鼠灌胃给药后均可吸收入血,而且口服吸收后在肝、肺、肾和血清中均有一定的分布。     

Improvement of in vivo stability of phosphodiester oligonucleotide using anionic liposomes in mice

Antisense phosphodiester oligonucleotides (ODN) are unstable in biological fluids due to nuclease-mediated degradation and therefore cannot be used in most antisense therapeutic applications. We describe here an in vitro and in vivo stabilization of a 15 mer phosphodiester sequence using anionic liposomes. Two formulations have been studied: DOPC/OA/CHOL and DOPE/OA/CHOL (pH-sensitive liposomes). Our in vitro findings reveal the same stabilization effect in mouse plasma for both anionic liposomes. In vivo investigation showed a great protective effect for both formulations after intravenous administration to mice. By contrast with in vitro results, a higher protection of ODN was observed with DOPC/OA/CHOL liposomes compared to the DOPE/OA/CHOL formulation. The latter was degraded in blood (75% of the injected dose at 5 min) probably due to interactions with blood components, and the remaining (25% at 5 min) was distributed mostly to the liver and spleen. DOPC liposomes were remarkably stable in blood and were distributed more slowly to all studied organs (liver, spleen, kidneys and lungs). Intact ODN was still observed in some organs (liver, spleen, lungs), but not in blood, 24 hours after DOPC liposome administration. These results suggest that this antisense strategy using carrier systems may be applicable to the treatment of diseases involving the reticuloendothelial system.     

Tuftsin-modified alginate nanoparticles as a noncondensing macrophage-targeted DNA delivery system

The main objective of this study was to evaluate macrophage-targeted alginate nanoparticles as a noncondensing gene delivery system for potential anti-inflammatory therapy. An external gelation method was employed to form plasmid DNA-encapsulated alginate nanoparticles. The nanoparticle surface was modified with a peptide sequence containing tuftsin (TKPR), and transfection efficiency was determined in J774A.1 macrophages. The effect of transfected mIL-10 in blocking expression of tumor necrosis factor-alpha (TNF-α) was evaluated in lipopolysaccharide (LPS)-stimulated cells. Scrambled peptide- and tuftsin-modified cross-linked alginate nanoparticles efficiently encapsulated plasmid DNA and protected against DNase I degradation. The transgene expression efficiencies, measured using GFP and mIL-10 expressing plasmid DNA, were highest with tuftsin-modified nanoparticles. Levels of TNF-α were significantly lower (p < 0.0001) in LPS-stimulated cells that were transfected with mIL-10 using alginate nanoparticles. The results of the study show that noncondensing alginate nanoparticles can efficiently deliver plasmid DNA, leading to sustained in vitro gene expression in macrophages.     

Morphological Analysis of Reticuloendothelial System in Capuchin Monkeys (Sapajus spp.) after Meso-2,3-Dimercaptosuccinic Acid (DMSA) Coated Magnetic Nanoparticles Administration

Magnetic nanoparticles can be used for numerous in vitro and in vivo applications. However, since uptake by the reticuloendothelial system represents an obstacle for the achievement of nanoparticle diagnostic and therapeutic goals, the aim of the present study was to evaluate the uptake of dimercaptosuccinic acid coated magnetic nanoparticles by reticuloendothelial system phagocytic cells present in lymph nodes, spleen, and liver tissue and how the presence of these particles could have an impact on the morphology of these organs in capuchin monkeys (Sapajus spp.). Animals were intravenously injected with dimercaptosuccinic acid coated magnetic nanoparticles and euthanized 12 hours and 90 days post-injection. Organs were processed by transmission electron microscopy and histological techniques. Samples of spleen and lymph nodes showed no morphological changes. Nevertheless, liver samples collected 90 days post-administration showed slight morphological alteration in space of Disse. Moreover, morphometrical analysis of hepatic mitochondria was performed, suggesting a clear positive correlation between mitochondrial area and dimercaptosuccinic acid coated magnetic nanoparticles administration time. The present results are directly relevant to current safety considerations in clinical diagnostic and therapeutic uses of magnetic nanoparticles.     

Incorporation of orotic acid into nucleotides and RNA in mouse organs during 60 minutes.

Mouse kidney and liver were found to increase their levels of radioactivity above that of serum from 2 to 60 min after administration of [6-14C]orotic acid. In spleen, thymus and brain, the radioactivity level reached a maximum soon after the injection and then decreased, as did that in serum. Sixty minutes after the injection, 44% of the administered isotope dose was found in the kidneys, 22% in the liver and 0.75% in the spleen. The 14C activity in liver UTP increased rapidly and then remained constant for 60 min. The ratio between the activities in uridine phosphates and UDP-sugars was 3:4 from 10- 60 min after injection. In the liver and kidneys, the RNA 14C activities at 60 min after injection were 15% of the activity in their acid-soluble fractions. Intraperitoneal administration was found to be preferable to intravenous administration for studies on nucleotides and RNA in mouse liver, due to the delayed incorporation of the [14C]orotic acid activity into the nucleotide pool.     

Acute Toxicity of Amorphous Silica Nanoparticles in Intravenously Exposed ICR Mice

This study aimed to evaluate the acute toxicity of intravenously administrated amorphous silica nanoparticles (SNPs) in mice. The lethal dose, 50 (LD₅₀), of intravenously administrated SNPs was calculated in mice using Dixon''s up-and-down method (262.45±33.78 mg/kg). The acute toxicity was evaluated at 14 d after intravenous injection of SNPs at 29.5, 103.5 and 177.5 mg/kg in mice. A silicon content analysis using ICP-OES found that SNPs mainly distributed in the resident macrophages of the liver (10.24%ID/g), spleen (34.78%ID/g) and lung (1.96%ID/g). TEM imaging showed only a small amount in the hepatocytes of the liver and in the capillary endothelial cells of the lung and kidney. The levels of serum LDH, AST and ALT were all elevated in the SNP treated groups. A histological examination showed lymphocytic infiltration, granuloma formation, and hydropic degeneration in liver hepatocytes; megakaryocyte hyperplasia in the spleen; and pneumonemia and pulmonary interstitial thickening in the lung of the SNP treated groups. A CD68 immunohistochemistry stain indicated SNPs induced macrophage proliferation in the liver and spleen. The results suggest injuries induced by the SNPs in the liver, spleen and lungs. Mononuclear phagocytic cells played an important role in the injury process.     

硅纳米颗粒作为基因转染载体的研究

通过不同浓度的NaCl、NaI修饰硅纳米颗粒,用琼脂糖凝胶电泳分析硅纳米颗粒与DNA结合力及对DNA的保护作用,同时用绿色荧光蛋白基因作报告基因,以硅纳米颗粒作为基因转染的载体,转染HT1080细胞。经电镜观察证实硅纳米颗粒进入细胞内;硅纳米颗粒与DNA结合后,能对DNA起保护作用;并且硅颗粒作为基因转染的载体,将绿色荧光蛋白基因导入HT1080细胞,用荧光显微镜观察到发绿色荧光的细胞。结果表明,硅纳米颗粒可作为基因转染的载体。     

Rapid Pharmacokinetic and Biodistribution Studies Using Cholorotoxin-Conjugated Iron Oxide Nanoparticles: A Novel Non-Radioactive Method

Background

Recent advances in nanotechnology have led to the development of biocompatible nanoparticles for in vivo molecular imaging and targeted therapy. Many nanoparticles have undesirable tissue distribution or unacceptably low serum half-lives. Pharmacokinetic (PK) and biodistribution studies can help inform decisions determining particle size, coatings, or other features early in nanoparticle development. Unfortunately, these studies are rarely done in a timely fashion because many nanotechnology labs lack the resources and expertise to synthesize radioactive nanoparticles and evaluate them in mice.

Methodology/Principal Findings

To address this problem, we developed an economical, radioactivity-free method for assessing serum half-life and tissue distribution of nanoparticles in mice. Iron oxide nanoparticles coated with chitosan and polyethylene glycol that utilize chlorotoxin as a targeting molecule have a serum half-life of 7–8 hours and the particles remain stable for extended periods of time in physiologic fluids and in vivo. Nanoparticles preferentially distribute to spleen and liver, presumably due to reticuloendothelial uptake. Other organs have very low levels of nanoparticles, which is ideal for imaging most cancers in the future. No acute toxicity was attributed to the nanoparticles.

Conclusions/Significance

We report here a simple near-infrared fluorescence based methodology to assess PK properties of nanoparticles in order to integrate pharmacokinetic data into early nanoparticle design and synthesis. The nanoparticles tested demonstrate properties that are excellent for future clinical imaging strategies and potentially suitable for targeted therapy.     

Role of organ-associated NK cells in decreased formation of experimental metastases in lung and liver

Mice treated with anti-asialo GM1 (asGM1) serum exhibited increased formation of experimental metastases in lung and liver after i.v. challenge with B16 melanoma or Lewis lung carcinoma. This increased metastasis formation coincided with decreased splenic NK activity and increased survival of i.v. injected radiolabeled tumor cells. In contrast, the injection of mice with the pyran copolymer maleic anhydride divinyl ether (MVE-2) augmented NK activity in the spleen and significantly depressed the formation of experimental metastases in the lungs and liver. However, a single or double administration of anti-asGM1 antiserum to MVE-2-pretreated mice failed to inhibit the immunoprophylaxis associated with MVE-2 administration, although it did decrease splenic NK activity and also increased the survival of i.v.-injected radiolabeled tumor cells. To address the mechanism for this dichotomy, we examined NK activity not only in the spleen but also in the blood, lungs, and livers of MVE-2-treated mice. Levels of NK activity in the lungs and liver were several-fold higher than those observed in spleen and blood. However, MVE-2-augmented NK activity in lung and liver was more resistant to depletion by the standard regimen of anti-asGM1 treatment than was NK activity in blood and spleen, and required two high-dose administrations of a higher titered antiserum for depletion of the augmented response. This high-dose regimen removed all detectable NK activity from the lung and liver, and concomitantly eliminated the metastasis-inhibiting effect of MVE-2. These data are consistent with a role for organ-associated NK cells in inhibiting metastasis formation during the extravasation and/or early postextravasation phases of the metastatic process. The results also suggest that biologic effects of NK activity in spleen and blood can be dissociated from those mediated by NK activity in other organs by use of different treatment regimens with anti-asGM1 serum. Finally, because NK activity in target organs can be augmented to an even greater extent than in the blood and spleen by at least some biologic response modifiers (BRMs), organ-associated NK activity should be considered as a possible mechanism for the therapeutic effects of BRM treatment.     

Studies on the oridonin-loaded poly(D,L-lactic acid) nanoparticles in vitro and in vivo

The purpose of this paper was to investigate the possibility of developing a polymeric nanoparticle delivery system for ORI to increase its solubility, blood circulation time and tissue targeting. Oridonin-loaded poly(D,L-lactic acid) nanoparticles (ORI-PLA-NP) were prepared by the further modified spontaneous emulsion solvent diffusion (MSESD) method. Studies were carried out to characterize and evaluate the produced ORI-PLA-NP both in vitro and in vivo. The experimental results showed that the mean size of the nanoparticles were 137.3 nm, with 87.2% of the nanoparticles distributed between the range of 107 and 195 nm. The entrapment efficiency and actual drug loading of the nanoparticles were 91.88+/-1.83 and 2.32+/-0.05%, respectively. It was demonstrated by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD) that ORI existed in the form of amorphous in the nanoparticles. The in vitro release profile of ORI-PLA-NP could be expressed well by the Higuchi equation: Q=8.944t(1/2)+11.246. The results of pharmacokinetics demonstrated that being encapsulated in PLA nanoparticles was remarkably effective for ORI to prolong its blood circulation time. After the i.v. administration of ORI-PLA-NP, we could observe a stable and high concentration of ORI in liver, lung and spleen, while its distribution in heart and kidney decreased.     

Tat peptide directs enhanced clearance and hepatic permeability of magnetic nanoparticles

Superparamagnetic nanoparticles have a number of important biomedical applications, serving as MR contrast agents for imaging specific molecular targets, as reagents for cell labeling and cell tracking, and for the isolation of specific classes of cells. We have determined the physical and biological properties of MION-47 and amino-CLIO, nanoparticles which serve as precursors for the synthesis of targeted MR contrast agents, and Tat-CLIO, a nanoparticle used as a cell labeling reagent. Blood half-lives for MION-47 and amino-CLIO were 682 +/- 34 and 655 +/- 37 min, respectively. The attachment of 9.7 tat peptides per crystal to amino-CLIO resulted in a reduction in blood half-life to 47 +/- 6 min. MION-47, amino-CLIO, and Tat-CLIO were present in highest concentrations in liver and spleen and lymph nodes, where concentrations for all three nanoparticles ranged from 8.80 to 6.11% of injected dose per gram. Twenty-four hours after the intravenous injection of amino-CLIO, the nanoparticle was concentrated in cells surrounding hepatic blood vessels (endothelial and Kupffer cells), in a fashion similar to that obtained with other nanoparticle preparations. In contrast, Tat-CLIO was present as numerous discrete foci of intense fluorescence throughout the parenchyma. Using the peptide as a component of future nanoparticles, it might be possible to design sensors for the detection of macromolecules present in intracellular compartments.     

Respiratory Syncytial Virus Fusion Glycoprotein Expressed in Insect Cells Form Protein Nanoparticles That Induce Protective Immunity in Cotton Rats

Respiratory Syncytial Virus (RSV) is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F) surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.     

In vitro evaluation of nanoparticles spleen capture

After intravenous injection, the main part of nanoparticles trapped by the spleen are concentrated in the marginal zone. The first step of this capture is the adhesion of the particles to the marginal zone macrophages. As classical techniques of cell suspension preparation did not allow to isolate without damage these actively capturing cells, tightly bound to a well-developed reticular meshwork, we designed a tissue slice incubation method, in order to study in vitro the interaction of nanoparticles with these particular macrophages, in conditions close to in vivo. In a serum supplemented medium, this in vitro model was able to give similar uptake profile than after intravenous injection of nanoparticles thus proving its validity. Surprisingly, no significant decrease of nanoparticles capture was observed when the medium was depleted from complement, immunoglobulins or proteins affine for heparin, while substitution of serum by purified albumin allowed a near optimal uptake. Addition of competitive ligands for lectin-like receptors did not show any clear inhibition of spleen capture. On the other hand, the scavenger receptor blocking agents, such as maleylated albumin or polyinosinic acid, induced a strong reduction of the spleen nanoparticles uptake. Thus, this paper proposes an in vitro binding assay as a reliable method to investigate the spleen capture of a large variety of nanoparticulate drug carriers. It is also a useful methodology to highlight the interactions between spleen cells and nanoparticles. The data obtained suggest that capture of nanoparticles depends on a multifactorial and complex phenomenon involving for a part albumin and the scavenger receptor.     

Nanoparticles for drug delivery to the lungs

The lungs are an attractive route for non-invasive drug delivery with advantages for both systemic and local applications. Incorporating therapeutics with polymeric nanoparticles offers additional degrees of manipulation for delivery systems, providing sustained release and the ability to target specific cells and organs. However, nanoparticle delivery to the lungs has many challenges including formulation instability due to particle-particle interactions and poor delivery efficiency due to exhalation of low-inertia nanoparticles. Thus, novel methods formulating nanoparticles into the form of micron-scale dry powders have been developed. These carrier particles exhibit improved handling and delivery, while releasing nanoparticles upon deposition in the lungs. This review covers the development of nanoparticle formulations for pulmonary delivery as both individual nanoparticles and encapsulated within carrier particles.     

Fate of intravenously administered rat lymphokine-activated killer cells labeled with different markers

Summary Rat lymphokine-activated killer (LAK) cells, generated by adhering rat splenocytes isolated from the 52% Percoll density fraction to plastic flasks, demonstrate restricted in vivo tissue distribution, localizing in the lungs and liver after 2 h, but redistributing into the liver and spleen 24 h after i.v. administration. However, a different pattern of distribution was observed when this population of LAK cells was labeled with one of four commonly used radioisotopes. For example, LAK cells showed a high distribution into the lungs 30 min after administration when labeled with⁵¹Cr,¹²⁵I-dUrd or¹¹¹In-oxine, whereas¹¹¹InCl-labeled LAK cells showed an equal distribution into the blood, lungs and liver at this time. Two hours after administration, cells labeled with¹¹¹In-oxine showed an equivalent distribution into the lungs and liver, those labeled with¹²⁵I-dUrd or⁵¹Cr showed a high accumulation in the lungs, whereas those labeled with¹¹¹In-Cl entered more into the liver and blood. The pattern of distribution of¹¹¹In-Cl- or¹¹¹In-oxine-labeled cells was confirmed using gamma camera imaging analysis. By 24 h, LAK cells labeled with¹¹¹InCl,¹¹¹In-oxine or⁵¹Cr distributed in the liver and spleen in variable concentrations. In contrast, cells labeled with¹²⁵I-dUrd were not detected in any organ tested.This study was paralleled by monitoring the distribution of LAK cells labeled with Hoechst 33342 (H33342) and analyzed for the presence of fluoresceinated cells in different organs either by flow cytometry analysis, or in frozen section. The data indicate that the distribution pattern of LAK cells labeled with¹¹¹In-oxine is the closest to the distribution of H33342-labeled cells. Of all the radioisotopes used,¹²⁵I-dUrd has the most disadvantages and is not recommended for monitoring the in vivo distribution of leukocytes.