Agglomerates Containing Pantoprazole Microparticles: Modulating the Drug Release

Pantoprazole-loaded microparticles were prepared using a blend of Eudragit® S100 and Methocel® F4M. The accelerated stability was carried out during 6 months at 40°C and 75% relative humidity. In order to improve technological characteristics of the pantoprazole-loaded microparticles, soft agglomerates were prepared viewing an oral delayed release and gastro-resistant solid dosage form. The agglomeration was performed by mixing the pantoprazole microparticles with spray-dried mannitol/lecithin powders. The effects of factors such as the amount of lecithin in the spray-dried mannitol/lecithin powders and the ratio between pantoprazole microparticles and spray-dried mannitol/lecithin powders were evaluated. The pantoprazole-loaded microparticles present no significant degradation in 6 months. The agglomerates presented spherical shape, with smooth surface and very small quantity of non-agglomerated particles. The agglomerates presented different yields (35.5–79.0%), drug loading (58–101%), and mechanical properties (tensile strength varied from 44 to 69 mN mm⁻²), when the spray-dried mannitol/lecithin powders with different lecithin amounts were used. The biopharmaceutical characteristics of pantoprazole microparticles, i.e., their delayed-release properties, were not affected by the agglomeration process. The gastro-resistance of the agglomerates was affected by the amount of spray-dried mannitol/lecithin powders. The ratio of lecithin in the spray-dried mannitol/lecithin powders was the key factor in the agglomerate formation and in the drug release profiles. The agglomerates presenting better mechanical and biopharmaceutical characteristics were prepared with 1:2 (w/w) ratio of pantoprazole-loaded microparticles and mannitol/lecithin (80:20) powder.Key words: agglomerates, delayed release, gastro-resistance, microparticles     

Conjugation of polyamidoamine dendrimers on biodegradable microparticles for nonviral gene delivery

We report on the preparation and characterization of poly(D, L-lactide-co-glycolide) (PLGA) microparticles with surface-conjugated polyamidoamine (PAMAM) dendrimers of varying generations. The buffering capacity and zeta-potential of the PLGA PAMAM microparticles increased with increasing generation level of the PAMAM dendrimer conjugated. Conjugation of the PAMAM dendrimer to the surface of the PLGA microparticle removed generation-dependent cytotoxicity in HEK293 and COS7 cell lines. PLGA PAMAM pDNA microparticles displayed similar cytotoxicity profiles to unmodified PLGA pDNA microparticles in COS7 cells. A generation three PAMAM dendrimer conjugated to PLGA microparticles significantly increased transfection efficiencies in comparison to unmodified PLGA microparticles.     

Development of Budesonide Microparticles Using Spray-Drying Technology for Pulmonary Administration: Design, Characterization, In Vitro Evaluation, and In Vivo Efficacy Study

The purpose of this research was to generate, characterize, and investigate the in vivo efficacy of budesonide (BUD) microparticles prepared by spray-drying technology with a potential application as carriers for pulmonary administration with sustained-release profile and improved respirable fraction. Microspheres and porous particles of chitosan (drug/chitosan, 1:2) were prepared by spray drying using optimized process parameters and were characterized for different physicochemical parameters. Mass median aerodynamic diameter and geometric standard deviation for conventional, microspheres, and porous particles formulations were 2.75, 4.60, and 4.30 μm and 2.56, 1.75, and 2.54, respectively. Pharmacokinetic study was performed in rats by intratracheal administration of either placebo or developed dry powder inhalation (DPI) formulation. Pharmacokinetic parameters were calculated (Ka, Ke, T _max, C _max, AUC, and Vd) and these results indicated that developed formulations extended half life compared to conventional formulation with onefold to fourfold improved local and systemic bioavailability. Estimates of relative bioavailability suggested that developed formulations have excellent lung deposition characteristics with extended T _1/2 from 9.4 to 14 h compared to conventional formulation. Anti-inflammatory activity of BUD and developed formulations was compared and found to be similar. Cytotoxicity was determined in A549 alveolar epithelial cell line and found to be not toxic. In vivo pulmonary deposition of developed conventional formulation was studied using gamma scintigraphy and results indicated potential in vitro–in vivo correlation in performance of conventional BUD DPI formulation. From the DPI formulation prepared with porous particles, the concentration of BUD increased fourfold in the lungs, indicating pulmonary targeting potential of developed formulations.     

Synthesis, cellular transport, and activity of polyamidoamine dendrimer-methylprednisolone conjugates

Dendrimers have emerged as promising multifunctional nanomaterials for drug delivery due to their well-defined size and tailorability. We compare two schemes to obtain methylprednisolone (MP)-polyamidoamine dendrimer (PAMAM-G4-OH) conjugate. Glutaric acid (GA) was used as a spacer to facilitate the conjugation. In scheme A, PAMAM-G4-OH was first coupled to GA and then further conjugated with MP to obtain PAMAM-G4-GA-MP conjugates. This scheme yields a lower conjugation ratio of MP, presumably because of lower reactivity and steric hindrance for the steroid at the crowded dendrimer periphery. In scheme B, this steric hindrance was overcome by first preparing the MP-GA conjugate, which was then coupled to the PAMAM-G4-OH dendrimer. The (1)H NMR spectrum of the conjugate from scheme B indicates a conjugation of 12 molecules of MP with the dendrimer, corresponding to a payload of 32 wt %. In addition, conjugates were further fluorescent-labeled with fluoroisothiocynate (FITC) to evaluate the dynamics of cellular entry. Flow cytometry and UV/visible spectroscopic analysis showed that the conjugate is rapidly taken up inside the cell. Fluorescence and confocal microscopy images on A549 human lung epithelial carcinoma cells treated with conjugates show that the conjugate is mostly localized in cytosol. MP-GA-dendrimer conjugate showed comparable pharmacological activity to free MP, as measured by inhibition of prostaglandin secretion. These conjugates can potentially be further conjugated with a targeting moiety to deliver the drugs to specific cells in vivo.     

Effects of structure of polyamidoamine dendrimer on gene transfer efficiency of the dendrimer conjugate with alpha-cyclodextrin

To improve gene transfer activity of a new nonviral vector, a polyamidoamine dendrimer (G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)), we prepared alpha-CDE conjugates with dendrimer having different generations (G3 and G4), and their gene transfer activities were compared with those of alpha-CDE conjugate (G2) and TransFast, a novel transfection reagent. alpha-CDE conjugates (G2, G3, and G4) formed the complexes with pDNA, changing the zeta-potential and particle size of pDNA complexes and the protection of pDNA from DNase I in a charge ratio-dependent manner, although their differences at higher charge ratios (vector/pDNA) were small. The gene transfer activity of alpha-CDE conjugates (G2, G3, and G4) was higher than that of the corresponding dendrimer alone in NIH3T3 and RAW264.7 cells. Of these CDE conjugates, alpha-CDE conjugate (G3) had a superior gene transfer activity which was comparable to that of TransFast in NIH3T3 cells. The intracellular distribution of pDNA after application of the pDNA complex with alpha-CDE conjugate (G3) to NIH3T3 cells was different from that with dendrimer alone (G3), although the cellular association of pDNA was almost comparable among all vectors. alpha-CDE conjugate (G3) strongly interacted with a fluorescence probe, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS), suggesting that the conjugate possesses the inclusion ability with biomembrane constituents such as phospholipids after transfection. These results suggest that alpha-CDE conjugates, particularly the G3 conjugate, could be novel nonviral gene transfer agents.     

<Emphasis Type="Italic">In Vivo</Emphasis> Anticancer Efficacy and Toxicity Studies of a Novel Polymer Conjugate <Emphasis Type="Italic">N</Emphasis>-Acetyl Glucosamine (NAG)–PEG–Doxorubicin for Targeted Cancer Therapy

A novel polymer–drug conjugate, polyethylene glycol–N-(acetyl)-glucosamine–doxorubicin (PEG-NAG-DOX) was evaluated in this study for its in vivo potential for treatment of tumours demonstrating improved efficacy and reduced toxicity. The proposed polymer–drug conjugate comprised of polyethylene glycol–maleimide (mPEG-MAL, 30000 Da) as a carrier, doxorubicin (DOX) as an anticancer drug and N-acetyl glucosamine (NAG) as a targeting moiety as well as penetration enhancer. Doxorubicin has a potent and promising anticancer activity; however, severe cardiotoxicity limits its application in cancer treatment. By modifying DOX in PEG-NAG-DOX prodrug conjugate, we aimed to eliminate this limitation. In vivo anticancer efficacy of the conjugate was evaluated using BDF mice-induced skin melanoma model by i.v. administration of DOX conjugates. Anticancer efficacy studies were done by comparing tumour volume, body weight, organ index and percent survival rate of the animals. Tumour suppression achieved by PEG-NAG-DOX at the cumulative dose of 7.5 mg/kg was two-fold better than that achieved by DOX solution. Also, the survival rate for PEG-NAG-DOX conjugate was >70% as compared to <50% survival rate for DOX solution. In addition, toxicity studies and histopathological studies revealed that while maintaining its cytotoxicity towards tumour cells, PEG-NAG-DOX conjugate showed no toxicities to major organs. Therefore, PEG-NAG-DOX conjugate can be suggested as a desirable candidate for targeted cancer therapy.     

PAMAM dendrimers for the delivery of the antibacterial Triclosan

Many oral care products incorporate an antibacterial compound to prevent the formation of dental plaque which predisposes teeth to dental caries or periodontal disease []. Triclosan (TCN) is a commonly used antiplaque agent in toothpastes []. Strategies to increase the delivery efficiency of antibacterials using formulation aids such as polyamidoamine (PAMAM) dendrimers are of interest.

Solubilisation studies over the pH range 5-12 demonstrated an increase in the level of TCN solubilised with increasing dendrimer concentration (1 mM–5 mM). However, the dendrimer was unable to enhance TCN solubility at lower pH values and the solubilising effect observed was attributed to the ionization of TCN (pKa 8.14) resulting from dendrimer induced pH changes.

End group modification of G3 PAMAM dendrimer with phenylalanine in order to promote solubility through π–π stacking between TCN and the amino acid has been carried out. Phenylalanine:G3 PAMAM conjugates of different ratios (32:1, 21:1, 16:1) were synthesized. The fully conjugated dendrimer (32:1) had poor aqueous solubility, whereas the 21:1 and 16:1 dendrimer conjugates were water soluble. The 21:1 conjugate was tested for its ability to solubilise TCN, however, again there was no increase over control buffer solutions of the same pH. An alternative approach under investigation is to directly conjugate TCN to PAMAM dendrimers via a hydrolysable linkage.     

Roller Compaction, Granulation and Capsule Product Dissolution of Drug Formulations Containing a Lactose or Mannitol Filler, Starch, and Talc

This study investigated the influence of excipient composition to the roller compaction and granulation characteristics of pharmaceutical formulations that were comprised of a spray-dried filler (lactose monohydrate or mannitol), pregelatinized starch, talc, magnesium stearate (1% w/w) and a ductile active pharmaceutical ingredient (25% w/w) using a mixed-level factorial design. The main and interaction effects of formulation variables (i.e., filler type, starch content, and talc content) to the response factors (i.e., solid fraction and tensile strength of ribbons, particle size, compressibility and flow of granules) were analyzed using multi-linear stepwise regression analysis. Experimental results indicated that roller compacted ribbons of both lactose and mannitol formulations had similar tensile strength. However, resulting lactose-based granules were finer than the mannitol-based granules because of the brittleness of lactose compared to mannitol. Due to the poor compressiblility of starch, increasing starch content in the formulation from 0% to 20% w/w led to reduction in ribbon solid fraction by 10%, ribbon tensile strength by 60%, and granule size by 30%. Granules containing lactose or more starch showed less cohesive flow than granules containing mannitol and less starch. Increasing talc content from 0% to 5% w/w had little effect to most physical properties of ribbons and granules while the flow of mannitol-based granules was found improved. Finally, it was observed that stored at 40 °C/75% RH over 12 weeks, gelatin capsules containing lactose-based granules had reduced dissolution rates due to pellicle formation inside capsule shells, while capsules containing mannitol-based granules remained immediate dissolution without noticeable pellicle formation.     

Tumor cell imaging using the intrinsic emission from PAMAM dendrimer: a case study with HeLa cells

HeLa 229 cells were treated with methotrexate (MTX) and doxorubicin (DOX), utilizing fourth generation (G4), amine terminated poly(amidoamine) {PAMAM} dendrimer as the drug carrier. In vitro kinetic studies of the release of both MTX and DOX in presence and absence of G4, amine terminated PAMAM dendrimers suggest that controlled drug release can be achieved in presence of the dendrimers. The cytotoxicity studies indicated improved cell death by dendrimer-drug combination, compared to the control experiments with dendrimer or drug alone at identical experimental conditions. Furthermore, HeLa 229 cells were imaged for the first time utilizing the intrinsic emission from the PAMAM dendrimers and drugs, without incorporating any conventional fluorophores. Experimental results collectively suggest that the decreased rate of drug efflux in presence of relatively large sized PAMAM dendrimers generates high local concentration of the dendrimer-drug combination inside the cell, which renders an easy way to image cell lines utilizing the intrinsic emission properties of PAMAM dendrimer and encapsulated drug molecule.     

Interaction of liposomes bearing a lipophilic doxorubicin prodrug with tumor cells

When used as nanosized carriers, liposomes enable targeted delivery and decrease systemic toxicity of antitumor agents significantly. However, slow unloading of liposomes inside cells diminishes the treatment efficiency. The problem could be overcome by the adoption of lipophilic prodrugs tailored for incorporation into lipid bilayer of liposomes. We prepared liposomes of egg yolk phosphatidylcholine and yeast phosphatidylinositol bearing a diglyceride conjugate of an antitumor antibiotic doxorubicin (a lipophilic prodrug, DOX-DG) in the membrane to study how these formulations interact with tumor cells. We also prepared liposomes of rigid bilayer-forming lipids, such as a mixture of dipalmitoylphosphatidylcholine and cholesterol, bearing DOX in the inner water volume, both pegylated (with polyethylene glycol (PEG) chains exposed to water phase) and non-pegylated. Efficiency of binding of free and liposomal doxorubicin with tumor cells was evaluated in vitro using spectrofluorimetry of cell extracts and flow cytometry. Intracellular traffic of the formulations was investigated by confocal microscopy; co-localization of DOX fluorescence with organelle trackers was estimated. All liposomal formulations of DOX were shown to distribute to organelles retarding its transport to nucleus. Intracellular distribution of liposomal DOX depended on liposome structure and pegylation. We conclude that the most probable mechanism of the lipophilic prodrug penetration into a cell is liposome-mediated endosomal pathway.     

Preparation and In Vitro Aerosol Performance of Spray-Dried Shuang-Huang-Lian Corrugated Particles in Carrier-Based Dry Powder Inhalers

The development of dry powder inhalation (DPI) products of traditional Chinese medicine (TCM) remains to be a challenge due to chemical complexity and batch-to-batch variations in constituent composition. This study was to investigate the feasibility of using spray-dried corrugated particles to improve the aerodynamic performance of a TCM, Shuang-Huang-Lian (SHL), in carrier-based DPI. Particles with different surface roughness were spray-dried by the addition of leucine and concomitant manipulation of spray-drying parameters. The surface roughness was determined by atomic force microscopy, whilst the aerodynamic performance of drug particle–mannitol/lactose blends was evaluated using a next-generation pharmaceutical impactor through a Cyclohaler. Although the emission efficiency for corrugated particle-based DPI was ∼10% lower than that for smooth SHL, the fine particle fractions (FPF_<4.4 μm) of 32.4–36.8% for the former were significantly higher than those of 14.7–16.2% for the latter. In particular, the FPF and fraction of drug detached from the carrier appeared not to be significantly affected by the variation in constituent composition of SHL. This study demonstrates that the use of corrugated particles in carrier-based DPI improved aerosol performance by facilitating drug detachment from the carrier, independent of variation in constituent composition, and such particles were potentially applicable to the development of SHL DPI products.KEY WORDS: dry powder inhaler, Shuang-Huang-Lian, spray-drying, surface roughness, traditional Chinese medicine     

Review on the targeted conjugation of anticancer drugs doxorubicin and tamoxifen with synthetic polymers for drug delivery

In this review, the binding and loading efficacy (LE) of anticancer drugs doxorubicin (DOX), tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox) with several synthetic polymers poly(ethylene glycol) (PEG), methoxypoly (ethylene glycol) polyamidoamine (mPEG-PAMAM-G3), and polyamidoamine (PAMAM-G4) dendrimers were compared in aqueous solution at pH 7.4. The results of multiple spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling of conjugated drug–polymer were examined. Structural analysis showed that drug–polymer conjugation occurs mainly via H-bonding and hydrophobic contacts. The order of binding is PAMAM-G4 > mPEG-PAMAM-G3 > PEG-6000 with 4-hydroxttamoxifen forming more stable conjugate than tamoxifen and endoxifen. Doxorubicin shows stronger affinity for PAMAM-G4 than tamoxifen and its metabolites. The drug LE was 30–55%. TEM showed significant changes in the carrier morphology upon drug encapsulation. Modeling also showed that drug is located in the surface and in the internal cavities of PAMAM with DOX forming more stable polymer conjugates.     

Thrombin-induced platelet microparticles improved the aggregability of cryopreserved platelets

Platelets were activated with freezing/thawing and thrombin stimulation, and platelet microparticles generated following platelet activation were isolated with ultracentrifugation. The effects of platelet microparticles on platelet activation were studied with annexin V assay, protein tyrosine phosphorylation, and platelet aggregation. Freezing-induced platelet microparticles decreased but thrombin-induced platelet microparticles increased platelet annexin V binding and aggregation. Freshly washed platelets were cryopreserved using epinephrine and dimethyl sulfoxide (Me(2)SO) as combined cryoprotectants, and stimulated with thrombin-induced platelet microparticles. Following incubation of thrombin-induced platelet microparticles, the reaction time of platelets to agonists decreased but the percentages of aggregation increased, such as washed platelets from 44% +/- 30 to 92% +/- 7, p < 0.001, and cryopreserved platelets from 66% +/- 10 to 77% +/- 7, p < 0.02. By increasing platelet aggregability, platelet microparticles recovered after thrombin stimulation improved platelet function for transfusion. A 53-kDa platelet microparticle protein showed little phosphorylation if it was released from resting platelets or platelets stimulated with ADP, epinephrine, propyl gallate or dephosphorylation if it was derived from ionophore A 23187-stimulated platelets. However, the same protein released from frozen platelets showed significant tyrosine phosphorylation. Since a microparticle protein with 53 kDa was compatible with protein tyrosine phosphatase-1B (PTP-1B), its phosphorylation suggests the inhibition of enzyme activity. The microparticle proteins derived from thrombin-stimulated platelets were significantly phosphorylated at 64 kDa and pp60c-src, suggesting that the activation of tyrosine kinases represents a possible mechanism of thrombin-induced platelet microparticles to improve platelet aggregation.     

Controlling filamentous fungi morphology with microparticles to enhanced β-mannanase production

β-mannanase was produced mainly by Aspergillus species and can degrade the β-1,4-mannose linkages of galactomannans. This study was undertaken to enhance mannanase production using talcum and aluminum oxide as the microparticles, which control cell morphology of recombinant Aspergillus sojae in glucose and carob extract medium. Both microparticles improved fungal growth in glucose and carob pod extract medium. Aluminum oxide (1 g/L) was the best agent for glucose medium which resulted in 514.0 U/ml. However, the highest mannanase activity was found as 568.7 U/ml with 5 g/L of talcum in carob extract medium. Increase in microparticle concentration resulted in decreasing the pellet size diameter. Furthermore, more than 10 g/L of talcum addition changed the filamentous fungi growth type from pellet to pellet/mycelium mixture. Results showed that right type and concentration of microparticle in fermentation media improved the mannanase activity and production rate by controlling the growth morphology.     

Microparticle adhesive dynamics and rolling mediated by selectin-specific antibodies under flow

In vitro studies were performed to characterize the relative performance of candidate receptors to target microparticles to inflammatory markers on vascular endothelium. To model the interactions of drug-bearing microparticles or imaging contrast agents with the vasculature, 6 micron polystyrene particles bearing antibodies, peptides, or carbohydrates were perfused over immobilized E- or P-selectin in a flow chamber. Microparticles conjugated with HuEP5C7.g2 (HuEP), a monoclonal antibody (mAb) specific to E- and P-selectin, supported leukocyte-like rolling and transient adhesion at venular shear rates. In contrast, microparticles conjugated with a higher affinity mAb specific for P-selectin (G1) were unable to form bonds at venular flow rates. When both HuEP and G1 were conjugated to the microparticle, HuEP supported binding to P-selectin in flow which allowed G1 to form bonds leading to stable adhesion. While the microparticle attachment and rolling performance was not as stable as that mediated by the natural ligands P-selectin Glycoprotein Ligand-1 or sialyl Lewis(x), HuEP performed significantly better than any previously characterized mAb in terms of mediating microparticle binding under flow conditions. HuEP may be a viable alternative to natural ligands to selectins for targeting particles to inflamed endothelium.     

Agglomerated Oral Dosage Forms of Artemisinin/β-Cyclodextrin Spray-Dried Primary Microparticles Showing Increased Dissolution Rate and Bioavailability

Artemisinin, a poorly water-soluble antimalarial drug, presents a low and erratic bioavailability upon oral administration. The aim of this work was to study an agglomerated powder dosage form for oral administration of artemisinin based on the artemisinin/β-cyclodextrin primary microparticles. These primary microparticles were prepared by spray-drying a water–methanol solution of artemisinin/β-cyclodextrin. β-Cyclodextrin in spray-dried microparticles increased artemisinin water apparent solubility approximately sixfold. The thermal analysis evidenced a reduction in the enthalpy value associated with drug melting, due to the decrease in drug crystallinity. The latter was also evidenced by powder X-ray diffraction analysis, while ¹³C-NMR analysis indicated the partial complexation with β-cyclodextrin. Agglomerates obtained by sieve vibration of spray-dried artemisinin/β-cyclodextrin primary microparticles exhibited free flowing and close packing properties compared with the non-flowing microparticulate powder. The in vitro dissolution rate determination of artemisinin from the agglomerates showed that in 10 min about 70% of drug was released from the agglomerates, whereas less than 10% of artemisinin was dissolved from raw material powder. Oral administration of agglomerates in rats yielded higher artemisinin plasma levels compared to those of pure drug. In the case of the agglomerated powder, a 3.2-fold increase in drug fraction absorbed was obtained.     

Preparation and characterization of poly(D,L-lactide-co-glycolide) microspheres for controlled release of human growth hormone

The purpose of this research was to assess the physicochemical properties of a controlled release formulation of recombinant human growth hormone (rHGH) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) composite microspheres. rHGH was loaded in poly(acryloyl hydroxyethyl) starch (acHES) microparticles, and then the protein-containing microparticles were encapsulated in the PLGA matrix by a solvent extraction/evaporation method. rHGH-loaded PLGA microspheres were also prepared using mannitol without the starch hydrogel microparticle microspheres for comparison. The detection of secondary structure changes in protein was investigated by using a Fourier Transfer Infrared (FTIR) technique. The composite microspheres were spherical in shape (44.6±2.47 μm), and the PLGA-mannitol microspheres were 39.7±2.50 μm. Drug-loading efficiency varied from 93.2% to 104%. The composite microspheres showed higher overall drug release than the PLGA/mannitol microspheres. FTIR analyses indicated good stability and structural integrity of HGH localized in the microspheres. The PLGA-acHES composite microsphere system could be useful for the controlled delivery of protein drugs.     

Application of a Four-fluid Nozzle Spray Drier to Prepare Inhalable Rifampicin-containing Mannitol Microparticles

The purpose of this study was to use a four-fluid nozzle spray drier as a new one-step method for preparing rifampicin (RFP)-containing mannitol microparticles. A RFP-acetone/methanol (2:1) solution and aqueous solutions of mannitol (MAN) were simultaneously supplied through different liquid passages of a four-fluid nozzle spray drier and then dried to obtain MAN microparticles containing RFP. Using a cascade impactor, the in vitro aerosol performance of RFP powder and RFP-MAN microparticles with 1:5, 1:10, and 1:20 ratios was compared. The in vivo retention of RFP in the lungs of rats after intratracheal administration of 1:20 RFP-MAN microparticles was also compared. The RFP-MAN microparticles had better aerosol performance than RFP powder and delivery to the lung stages improved as the fraction of MAN was increased. For the 1:20 RFP-MAN microparticles, deposition in stages 2–7 was approximately 43%, which is sufficient for treatment. Approximately 8% of the RFP-MAN microparticles were deposited in stages 6–7, which corresponds to alveoli containing alveolar macrophages. The initial retention of RFP in the lung following pulmonary delivery of 1:20 RFP-MAN microparticles was higher than following oral or intravenous administration of RFP, but the elimination was rapid, resulting in the disappearance of RFP from the lung within 4 h. The plasma concentration–time profile of RFP after intratracheal administration of 1:20 RFP-MAN microparticles was consistent with the profile for RFP retention in the lung. Addition of cholesterol or phosphatidylcholine to RFP had little effect on its retention in the lung. The RFP-MAN microparticles were effective for delivery of RFP to the lung, but the RFP rapidly removed from the lung into the blood circulation. This study demonstrated that RFP-containing MAN microparticles prepared in one step using the four-fluid nozzle spray drier efficiently deliver RFP to the lung, although methods must be developed to prolong its retention and improve targeting to alveolar macrophages.     

Formulation of the biocontrol agent <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> CPA-8 using different approaches: liquid,freeze-drying and fluid-bed spray-drying

The present work focuses on the assessment and comparison of three different formulation technologies and the effect of protectants on cell viability, storage stability and antagonistic activity of the biocontrol agent Bacillus amyloliquefaciens CPA-8. Cultures were concentrated with different protective substances such as MgSO₄, sucrose and skimmed milk (SM) and subjected to liquid formulation, freeze-drying and fluid-bed spray-drying. Results showed that CPA-8 freeze-dried cells without protectants or amended with SM suffered the highest losses in cell viability (0.41?0.48 log). Moreover, the cell viability of the tested freeze-dried products decreased after four months of storage at both tested temperatures (4 and 22 °C). Otherwise, liquid and fluid-bed spray-dried products were stable for four months at 4 °C and for 12 months at 22, 4 and ?20 °C, respectively, and no effect of the protectants was observed. The most suitable CPA-8 products were then tested against Monilinia laxa and M. fructicola in artificially wounded nectarines and in all cases the antagonistic activity was maintained similar to fresh cells. The efficacy results revealed that the formulation process did not affect the biocontrol potential of CPA-8. This work led us to conclude that effective formulations with final concentrations ranging from 1.93 × 10⁹–2.98 × 10⁹ CFU ml^?1 and from 4.76 × 10⁹–1.03 × 10¹⁰ CFU g^?1 were obtained for liquid and dried products, respectively. Additionally, the suitability of the fluid-bed spray drying technology should be taken into account to develop a stable and effective CPA-8 product for practical applications to control brown rot in stone fruit.     

Stepwise self-assembled poly(amidoamine) dendrimer and poly(styrenesulfonate) microcapsules as sustained delivery vehicles

Hollow microcapsules comprised of poly(styrenesulfonate) (PSS) and a fourth generation poly(amidoamine) dendrimer (4G PAMAM) were prepared by depositing PSS/4G PAMAM multilayers on melamine formaldehyde (MF) colloid particles by the layer-by-layer self-assembly technique and subsequently dissolving the templated cores. The PSS/4G PAMAM layers were unstable toward the core removal procedure (pH approximately 1), resulting in a low yield of intact hollow capsules (<10% for 3.5 microm diameter MF templates). Stretching of the multilayer film due to core swelling during MF core dissolution leads to partial or complete destruction of capsules, giving discrete PSS-4G PAMAM complexes. Yields were increased by increasing inter- and intramolecular attractive forces between the PSS chains in the capsules through electrostatic, hydrophobic, and a combination of these interactions. The yields, however, were practically unaffected by enhancing such effects between dendrimer molecules. Transmission electron microscopy and scanning force microscopy measurements show no deformation for 3.5 microm capsules stabilized through the various interactions stated above. Further, capsules were filled with low molecular weight dextran sulfate and subsequently loaded with a model, therapeutically active molecule, doxorubicin hydrochloride (DOX). Release of DOX from the capsules was also studied to highlight the drug delivery potential of the dendrimer-based microcapsules.