Two-photon confocal imaging study: cell uptake of two photon dyes-labeled PAMAM dendrons in HeLa cells

A two-photon excitation difluoroboron dye activated in the near infrared region for biological image analysis was synthesized in this study. Cell affinity, membrane interaction, and the endocytosis pathway of PAMAM dendrons were investigated using only covalent two-photon dyes (TPD) at the periphery of the PAMAM dendrons. Generation 3 TPD-labeled PAMAM dendrons (BG3) exhibited multivalency binding on the HeLa cell membranes from the cell affinity study in the fixation of HeLa cells. Photo-stimulation on the membrane of the living HeLa cell was observed by confocal optical imaging in situ, using the two-photon model, when incubated with BG3. Analyses of cell membrane integrity via lactate dehydrogenase (LDH) assay confirmed membrane damage at two photon excitation model. However, no variation in the cell was observed using the one-photon excitation model. These results indicated a high degree of dendrons uptake by cells through binding to the cell membrane following the endocytotic pathway. Furthermore, the wide excitation fluorescence spectrum of difluoroboron dye provides dual imaging with which to study the endocytosis of TPD-labeled PAMAM dendrons using a single near infrared laser.     

5-氟尿嘧啶缓释剂制备及体外释药性能比较*

背景：5-氟尿嘧啶-聚乳酸-乙醇酸共聚物缓释微球在青光眼滤过术后抑制滤过泡的瘢痕化具有潜在应用价值,但微球制备程序复杂,微球载药量一般较低,且药物突释现象明显。 目的：比较乳化溶剂挥发法制备的5-氟尿嘧啶-聚乳酸-乙醇酸共聚物微球和喷雾成膜法制备的5-氟尿嘧啶-聚乳酸-乙醇酸共聚物缓释膜两种缓释剂的形态、载药量、体外释放规律,以探讨获得缓释效果较佳的5-氟尿嘧啶缓释剂制备方法。 方法：以聚乳酸-乙醇酸共聚物为载体,采用乳化溶剂挥发法制备5-氟尿嘧啶-聚乳酸-乙醇酸共聚物微球;用喷雾成膜法制备5-氟尿嘧啶-聚乳酸-乙醇酸共聚物缓释膜。 结果与结论：用乳化溶剂挥发法制备的微球外观圆整,粒径为(4 447.4±359.8) nm,载药量(8.67±0.37)%,包封率为(86.68± 1.92)%;用喷雾成膜法制备的缓释膜表面光滑平整,质量为(13.76±0.26) mg ,直径为6 mm ,厚度为(0.24±0.005) mm,载药量(23.76±0.37)%,包封率为(95.04±1.36)%。缓释剂制备过程未影响5-氟尿嘧啶的药物性能。微球体外释放突释明显,缓释膜的体外释放平稳持久,释放曲线符合Higuchi方程。结果表明缓释膜制备方法更简单易行,且能明显提高缓释剂的载药量,降低突释现象,同时延长药物的缓释时间。 关键词：聚乳酸-聚乙醇酸;5-氟尿嘧啶;微球;缓释膜;体外释放 doi:10.3969/j.issn.1673-8225.2012.08.022     

PEG化PAMAM树状大分子的合成及其作为药物载体的研究

研究经聚乙二醇(PEG)进行表面修饰的聚酰胺-胺树枝状大分子作为药物载体时对药物的包裹及释放能力。用经三氟乙基磺酸单甲氧基(Tresylate)活化的相对分子质量为5000的单甲氧基聚乙二醇(Tresylated MPEG-5000)对聚酰胺-胺G4.0-PAMAM树状大分子修饰,目标产物PEG化树状大分子用FT-IR1、H NMR进行结构表征。MTT研究其细胞毒性。通过包裹与释放实验研究该药物载体对抗癌药物甲氨蝶呤(MTX)的包裹及释放能力。每个修饰后的树状大分子可包裹33个抗癌药物甲氨蝶呤分子,细胞毒指数较低,释药速度减慢。修饰后的树状大分子与未PEG化的树状大分子载体相比具有更强的包裹能力,并具有一定的药物缓释功能;细胞毒性试验表明,其胞毒性与未PEG化的树状大分子相比明显偏低。     

Reducing cytotoxicity while improving anti-cancer drug loading capacity of polypropylenimine dendrimers by surface acetylation

Polypropylenimine (PPI) dendrimers have been widely used as effective delivery vehicles for drugs and nucleic acids during the past decade. However, biomedical applications of PPI dendrimers were limited because of their serious cytotoxicity and low drug loading capacity. In the present study, acetylated PPI dendrimers with different degrees of acetylation ranging from 14.2% to 94.3% were synthesized and used to encapsulate drugs, including methotrexate sodium, sodium deoxycholate and doxorubicin. Acetylated PPI dendrimers with a degree of acetylation >80% showed a significantly decreased cytotoxicity (>90% cell viability) on MCF-7 and A549 cells. The drug loading capacity of acetylated PPI dendrimers increased proportionally with the degree of acetylation on the dendrimer surface. In addition, 94.3% acetylated PPI dendrimers exhibited a pH-responsive release profile of anticancer drugs loaded within the nanoparticles. The cytotoxicities of methotrexate sodium and doxorubicin on MCF-7 and A549 cells were significantly reduced when they were complexed with acetylated PPI dendrimers with high degrees of acetylation (>80%), owing to sustained drug release from the dendrimers. The results suggest that surface acetylation can reduce the cytotoxicity and improve the anticancer drug loading capacity of cationic dendrimers, and that acetylated PPI dendrimers are promising vehicles for anticancer drugs in clinical trials.     

Co-effect of aqueous solubility of drugs and glycolide monomer on in vitro release rates from poly(D,L-lactide-co-glycolide) discs and polymer degradation

The objective of this study was to investigate the effect of aqueous solubility of model drugs and glycolide monomer (GM) from poly(D,L-lactide-co-glycolide) (PLGA) discs on in vitro release rates and polymer degradation. 5-Fluorouracil (5-FU), a water-soluble compound, and dexamethasone in a water-insoluble base form were selected as model drugs. Glycolide monomer, that has moderate solubility in water, was a non-toxic and biodegradable additive as a derivative material of hydrolysis of PLGA in order to obtain desirable drugs release rates. PLGA discs with or without GM were formulated by means of compression molding method. The prepared polymeric discs were incubated at 37 degrees C in phosphate-buffered saline (PBS, pH 7.4) and characterized at scheduled time points for water uptake, mass loss, diameter and morphology change, molecular weight and composition change using scanning electron microscopy (SEM), gel-permeation chromatography (GPC), and H-NMR, respectively. The supernatants were taken out of the sample vials and were analyzed for drug release. The 5-FU release was found to be increasing in proportion to the drug loading amount with an initial burst for 5 days, while dexamethasone release showed inverse relationship with the increasing drug loading amount. However, the release behaviors of 5-FU and dexamethasone polymeric discs containing GM showed faster release rates than control discs (without GM) and did not show lag periods during the in vitro release test due to adding GM, which acted as a channeling agent that has moderate solubility in water. Polymer degradation was found to be affected by aqueous solubility of drugs and GM. In conclusion, we observed that drugs release rates were influenced by their aqueous solubility and loading amount and also GM plays a major role in controlling drug release rates regardless of solubility of drugs. This system appears to be promising for controlled drug delivery aimed at local therapy.     

Cell adhesion and proliferation on hydrophilic dendritically modified surfaces

Dendritically modified, or "dendronized" surfaces are generated by modification of a substrate with perfectly branched polymers, known as dendrimers. Here, such dendronized surfaces were prepared by initial chemisorption of poly(ethylene glycol)-mono-thiol (HS-PEG(650)-OH) onto gold-coated silicon wafers, followed by divergent synthesis of aliphatic polyester dendrons, generation 1-4, starting from the terminal PEG OH- group. The adhesion and proliferation of human corneal epithelial cells (HCEC) and mouse 3T3 fibroblasts (M-3T3) as model cells on these hydroxyl-terminated dendronized surfaces were investigated. In addition, the effect of covalently attaching PEG mono-methyl ether (PEG-OMe) chains (M(n)=2000Da) to the peripheral hydroxyl groups of G1- and G2-dendronized surfaces on adhesion and proliferation of the same cell lines was studied. Little or no HCEC adhesion was noted on gold surfaces modified with PEG mono-thiol (HO-PEG-SH) in serum-free medium. These cells showed a greater affinity for the dendronized surfaces compared to the control Au surfaces at early incubation stages (1 day). At longer incubation times, HCEC proliferation increased exponentially on the dendronized surfaces. However, when G1- and G2-dendronized surfaces were modified with PEG-OMe chains, adhesion of both HCEC and M-3T3 cells was significantly reduced. Cell studies with M-3T3 fibroblasts, carried out in serum-containing medium, showed that cell attachment was diminished for the PEG-grafted Au surfaces compared to the control Au and G1-G4 dendronized surfaces.     

In situ generation of sodium alginate/hydroxyapatite nanocomposite beads as drug-controlled release matrices

In order to find a new way to slow down the release of drugs and to solve the burst release problem of drugs from traditionally used hydrogel matrices, a series of novel pH-sensitive sodium alginate/hydroxyapatite (SA/HA) nanocomposite beads was prepared by the in situ generation of HA micro-particles in the beads during the sol–gel transition process of SA. The SA/HA nanocomposites were characterized by Fourier transform IR spectroscopy, X-ray fluorescence spectrometry, scanning electron microscopy and field emission SEM in order to reveal their composition and surface morphology as well as the role that the in situ generated HA micro-particles play. The factors influencing the swelling behavior, drug loading and controlled release behavior of the SA/HA nanocomposite beads were also investigated using diclofenac sodium (DS) as the model drug. The HA micro-particles act as inorganic crosslinkers in the nanocomposites, which could contract and restrict the movability of the SA polymer chains, and then change the surface morphology and decrease the swell ratio. Meanwhile, the entrapment efficiency of DS was improved, and the burst release of DS was overcome. The factors (including concentration of Ca²⁺, reaction time and temperature) affecting the growth of HA micro-particles have a clear influence on the entrapment efficiency and release rate of DS. In this work, the nanocomposite beads prepared under optimum condition could prolong the release of DS for 8 h more compared with the pristine SA hydrogel beads.     

Synthesis and characterization of dendritic star-shaped zwitterionic polymers as novel anticancer drug delivery carriers

In this work, a novel dendritic star-shaped zwitterionic polymer, polyamidoamine-graft-poly[3-dimethyl (methacryloyloxyethyl) ammonium propanesulfonate] (PAMAM-g-PDMAPS), was synthesized. PAMAM dendrimers (generation 2, G2) were firstly prepared and then converted into the PAMAM-Br macroinitiator with 2-bromoisobutyryl bromide for ATRP. Finally, ATRP of zwitterionic DMAPS was carried out to obtain the dendritic star-shaped polymers PAMAM-g-PDMAPS with different PDMAPS chain lengths. Fourier transform-infrared spectroscopy, ¹H?NMR, dynamic laser light scattering (DLS), and TEM were used to characterize the polymers. Encapsulation of adriamycin (ADR) by PAMAM-g-PDMAPS nanoparticles and ADR release behavior from ADR-loaded PAMAM-g-PDMAPS nanoparticles were investigated in detail. PAMAM-g-PDMAPS polymers, even starting from low-generation PAMAM core (G2), were found to show high loading efficiency for ADR because ADR existed not only within G2 PAMAM cores but also in PDMAPS layers. The release profile of ADR from ADR-loaded PAMAM-g-PDMAPS nanoparticles was pH-sensitive and could be controlled by the length of PDMAPS chains. Cell viability studies indicated that ADR-loaded PAMAM-g-PDMAPS could effectively restrain the growth of HepG2 cells and even kill them, whereas PAMAM-g-PDMAPS exhibited nontoxicity. All these results demonstrated that dendritic star-shaped zwitterionic polymers PAMAM-g-PDMAPS are attractive candidates as anticancer drug delivery carriers.     

Effect of biphasic calcium phosphates on drug release and biological and mechanical properties of poly(epsilon-caprolactone) composite membranes

Poly(epsilon-caprolactone) (PCL) and biphasic calcium phosphate (CaP) composite membranes were prepared for use in tissue regeneration by a novel solvent casting-pressing method. An antibiotic drug, tetracycline hydrochloride (TCH), was entrapped within the membranes to investigate the efficacy of the material as a drug delivery system. The CaP powders were varied in amount (0-50 wt %) and in powder characteristics by heat treating at different temperatures, and their effects on the mechanical and biological properties and drug release of the membranes were examined. With CaP addition up to 30 wt %, the elastic modulus of the membranes was enhanced much due to the rigidity of CaP. While the tensile strength and elongation rate decreased gradually with CaP addition because the CaP powders acted as a failure source. The osteoblast-like cells cultured on the CaP-PCL composite membranes exhibited significant improvements in proliferation and alkaline phosphatase (ALP) activity compared to pure PCL and culture plastic control, indicating excellent cell viability and functional activity. The TCH drugs were released from the PCL and CaP-PCL membranes in a similar fashion; an initial burst followed by a reduced release rate. The initial burst effect diminished much by the addition of CaP powders. The CaP addition increased the drug release rate after an initial period, and this was attributed to the high water uptake capacity and dissolution of the CaP containing membranes. Compared to the composite membranes containing heat-treated CaP powders, those with as-precipitated ones had higher dissolution and drug releases. These observations on mechanical properties and cellular responses as well as on drug release profiles suggested that the CaP-PCL composite membranes are potentially applicable to tissue regeneration and drug delivery system.     

Folate coupled poly(ethyleneglycol) conjugates of anionic poly(amidoamine) dendrimer for inflammatory tissue specific drug delivery

Folate receptor is overexpressed on the activated (but not quiescent) macrophages in both animal models and human patients with naturally occurring rheumatoid arthritis. The aim of this study was to prepare folate targeted poly(ethylene glycol) (PEG) conjugates of anionic dendrimer (G3.5 PAMAM) as targeted drug delivery systems to inflammation and to investigate its biodistribution pattern in arthritic rats. Folate-PEG-PAMAM conjugates, with different degrees of substitution were synthesized by a two-step reaction through a carbodiimide-mediated coupling reaction and loaded with indomethacin. Folate-PEG conjugation increased the drug loading efficiency by 10- to 20-fold and the in vitro release profile indicated controlled release of drug. The plasma pharmacokinetic parameters indicated an increased AUC, circulatory half-life and mean residence time for the folate-PEG conjugates. The tissue distribution studies revealed significantly lesser uptake by stomach for the folate-PEG conjugates, thereby limiting gastric-related side effect. The time-averaged relative drug exposure (r(e)) of the drug in paw for the folate-PEG conjugates ranged from 1.81 to 2.37. The overall drug targeting efficiency (T(e)) was highest for folate-PEG conjugate (3.44) when compared to native dendrimer (1.72). The folate-PEG-PAMAM conjugates are the ideal choice for targeted delivery of antiarthritic drugs to inflammation with reduced side-effects and higher targeting efficiency.     

Improving delivery of hydrophobic drugs from hydrogels through cyclodextrins

A simple and effective technique of improving delivery of hydrophobic drugs from swellable systems is presented. Conventional methods of drug loading in hydrogel systems are limited by the characteristics of the pharmacological agent. The approach we present uses complexants to modulate drug release. Crosslinked poly(ethylene glycol) (PEG) hydrogels were synthesized, characterized, and used for vascular applications. The release of cyclosporine (CyA) from PEG hydrogels is significantly altered by the sterilization techniques. It was hypothesized that the release of CyA from PEG hydrogels can be modulated by using complexants. A cyclodextrin-CyA complex solution was prepared and used for drug loading. The sterilized PEG hydrogels that were loaded using the cyclodextrin-CyA complex solution had favorable release characteristics compared with the release from PEG hydrogels that were loaded using the conventional technique. Hence, drug release from swellable systems can be tailored by the application of this strategy.     

Dendronized Hyperbranched Polyethyleneimines with Switchable Thermoresponsiveness and Encapsulation

Hyperbranched polyethyleneimines (PEIs) with multiple layered structures and high density of terminal amino groups have shown great potential for applications in biomedicine, sensors, and catalysis. In this work, a kind of dendronized hyperbranched PEIs (DHPs) is synthesized by modification of PEIs with dendritic oligoethylene glycols (OEGs). These DHPs not only inherit the characteristic thermoresponsive behavior from the OEG dendrons, but also show low cytotoxicity and switchable encapsulation capacities. Their thermoresponsive behavior is found to be mainly dependent on the coverage and amphiphilicity of dendritic OEGs, solution pH, and NaCl concentration. Moreover, DHPs exhibit advantages in thermally-switchable complexation with guest molecules because of their adjustable crowding effect from the densely packed OEG chains. In addition, these DHPs can also be used as nanoreactors, which can reduce metal ions into metal nanoparticles in a convenient way. In virtue of these peculiarities, it is believed that these dendronized hyperbranched PEIs with smart properties can be used as promising scaffolds in drug-controlled release and nanoreactors.     

Coaxial electrospun zein nanofibrous membrane for sustained release

Zein nanofibrous membranes for sustained release have been prepared by coaxial electrospinning. Core-sheath structure has been successfully fabricated using zein as both the core and sheath component. Impact of solvent and solution concentration on the morphology of the resulting fibers was investigated. Allyltriphenylphosphonium bromide was used as a model drug to test the sustained release effect. The sustained release profile and the antimicrobial activity of the resulting membranes were investigated and compared with that of the single fluid electrospinning of zein/drug blended membrane. The ratio of the inner and outer feeding rates was found to influence the encapsulation of drugs, and in turn affect the sustained release effect of the resulting membranes. The coaxial electrospinning membrane can remarkably suppress the initial burst release of drugs by giving a releasing amount of 15% in the first 1?h when the inner/outer ratio was larger than 1:2. This drug-loaded zein membrane with preferable sustained release effect can be applied in many fields such as wound healing and packaging sector.     

Cell proliferation and controlled drug release studies of nanohybrids based on chitosan-g-lactic acid and montmorillonite

The present paper reveals the potential uses of novel hybrids of chitosan-g-lactic acid and sodium montmorillonite (MMT) in controlled drug delivery and tissue engineering applications. The drug-loaded novel nanohybrid films and porous scaffolds have been prepared by solvent casting and freeze-drying of the grafted polymer solution, respectively. Sodium Ibuprofen was loaded into nanohybrids of chitosan-g-lactic acid/sodium montmorillonite (CS-g-LA/MMT). Grafting of lactic acid and the drug loading were characterized by Fourier transform infrared spectroscopy. Formation of intercalated nanocomposites was confirmed by X-ray diffraction. Mechanical properties measurements have shown improvement in modulus and strength with expense of elongation by MMT reinforcement. The nanohybrids were found to be stable regardless of pH of the medium. The cell proliferation profile also shows that prepared nanohybrids are biocompatible. MMT reinforcement was found to control the drug (Ibuprofen) release rate in phosphate buffer saline solution (pH 7.4). MMT clay is therefore a viable additive for formulating sustained drug delivery systems based on lactic acid grafted chitosan.     

Effect of Enzyme Modification by Well-Defined Multi-Armed Poly(Ethylene Glycol) Synthesized Using Polyamidoamine Dendron

Egg white lysozyme was chemically modified by PEGylated PAMAM 1st, 2nd and 3rd generation dendrons, which had 2, 4 and 8 PEG arms, respectively. The number of PEG chains introduced to the lysozyme molecule drastically increased with an increase in dendron generation, although the number of PEGylated PAMAM dendrons introduced decreased due to steric repulsion. The lytic activity of lysozyme to Micrococcus luteus cells was effectively inhibited by conjugating PEGylated PAMAM dendron to the lysozyme, indicating steric stabilization of PEG chains at the surface of lysozyme molecule. In addition, the enzymatic reaction of the lysozyme with oligosaccharide substrate was apparently accelerated by a substrate condensation effect due to the multi-armed structure of PEG.     

Insoluble ionic complexes of polyacrylic acid with a cationic drug for use as a mucoadhesive, ophthalmic drug delivery system

We have developed a new mucoadhesive drug delivery formulation based on an ionic complex of partially neutralized poly(acrylic acid) (PAA) and a highly potent beta blocker drug, levobetaxolol x hydrochloride (LB x HCl), for use in the treatment of glaucoma. PAA was neutralized with sodium hydroxide to varying degrees of neutralization. Aqueous solutions containing concentrations of LB x HCl equivalent to the degree of PAA neutralization were added to the PAA solutions and formed insoluble complexes, which were isolated. The complex formation was followed by turbidimetric titration, and the complexes were characterized by IR and 1H NMR spectroscopy. Complexes were prepared with varying degrees of drug loading, such that the same PAA chain would have free -COOH groups for mucoadhesion along with ionic complexes of LB x H+ with COO- groups. Thin films of the complexes dissociated to release the drug by ion exchange with synthetic tear fluid. The films shrunk continuously during release of the drug and dissolved completely in 1 h. Solid inserts of these films could be useful as a mucoadhesive ophthalmic drug delivery system.     

New hydrogel matrices containing an anti-inflammatory agent. Evaluation of in vitro release and photoprotective activity.

In the present work. the preparation and characterization of hydrogels based on alpha,beta-polyaspartylhydrazide (PAHy) chemically crosslinked with ethyleneglycol diglycidylether (EGDGE) containing Tolmetin sodium salt, are reported. In particular, these samples have been prepared both as water swellable microparticles and as gels at two different crosslinking degrees. The incorporation of Tolmetin sodium salt in PAHy-EGDGE microparticles has been performed after the crosslinking reaction by a soaking procedure or during the formation of the network. The influence of drug loading procedure on Tolmetin release has been evaluated by performing in vitro release study in simulated gastrointestinal fluids (pH 1.0/6.8) using the pH variation method and in phosphate buffered saline, pH 7.4. PAHy-EGDGE networks containing Tolmetin sodium salt have been also prepared as gels. These have showed a slowed down release as evidenced by in vitro release studies at pH 5.0 and 7.4 using a Franz diffusion cell system and an artificial membrane. Finally, PAHy EGDGE networks provide a pronounced reduction of the photosensitizing activity of Tolmetin, as evidenced by in vitro hemolysis assays.     

磁性聚乳酸-羟基乙酸氧化苦参碱纳米粒的制备及其特性

目的研究磁性聚乳酸-羟基乙酸氧化苦参碱纳米粒(M-PLGA-OM-NP)的制备工艺,并对纳米粒子进行评价。方法运用复乳法制备M-PLGA-OM-NP,通过透射电子显微镜观察纳米粒形态,并对纳米粒的平均粒径、载药量、包封率、体外释药情况等进行评价。结果纳米粒外观呈规则球形,其平均粒径为146.5 nm,载药量为7.61%,包封率为44.8%。突释后至第72小时,纳米粒维持较稳定的释药速度,累积释放达52.9%。72～240 h,药物释放缓慢,累计释放约为16.6%,体外释放符合Ritger peppas方程lny=1.280 6+lnt。氧化苦参碱药性不受温度影响。结论获得了较满意的M-PLGA-OM-NP制备工艺,其过程简单,粒子性状符合要求。     

9-硝基喜树碱叶酸聚合物胶束肿瘤靶向给药系统的表征及体外药效评价

背景：近年来,两亲性聚合物胶束作为难溶性药物载体和叶酸介导的肿瘤细胞靶向给药系统在药剂学研究领域受到极大的关注。 目的：制备包载9-硝基喜树碱的叶酸聚合物胶束并进行理化表征及体外药效评价。 方法：采用薄膜-水化法制得载药胶束,利用激光粒度分析仪检测胶束粒径大小,反相高效液相层析法检测载药量,透析法进行体外释放试验;利用肿瘤细胞摄取及体外生长抑制试验,对叶酸聚合物胶束作体外药效评价。 结果与结论：制得的9-硝基喜树碱叶酸聚合物胶束粒径为24~26 nm,载药量为3.24%,24 h累积释放百分率约90%。叶酸修饰的聚合物胶束对肿瘤细胞的亲和性及抗肿瘤活性显著高于普通胶束。提示叶酸修饰的聚合物胶束可为难溶性药物提供一种具有良好应用前景的肿瘤主动靶向纳米载药系统。     

Research on preparation and quality control of drug-carried poly lactic-co-glycolic acid microbubble

This research was aimed to develop the technique and formula for the preparation of stable and effective microbubbles containing hydrophilic drugs. We prepared EB-PLGA microbubbles and evaluated its drug loading and burst release to choose the best technique and formula. The result of optimizing formula was W1/O (1:15), EB-PLGA (0.04), PVA (5%). The burst release decreased after the addition of supplemental agent and the change of method for preparation. We concluded that the optimizing formula could elevate drug loading and decrease burst release obviously.