Microencapsulation of Lactobacillus plantarum (mtcc 5422) by spray-freeze-drying method and evaluation of survival in simulated gastrointestinal conditions

Spray-drying (SD) and freeze-drying (FD) are widely used methods for microencapsulation of heat-sensitive materials like probiotics for long-term preservation and transport. Spray-freeze-drying (SFD) is relatively a new technique that involves spraying a solution into a cold medium and removal of solvent (water) by conventional vacuum FD method. In this study, the SFD microencapsulated Lactobacillus plantarum powder (1:1 and 1:1.5 core-to-wall ratios of whey protein) is compared with the microencapsulated powders produced by FD and SD methods. The SFD and FD processed microencapsulated powder show 20% higher cell viability than the SD samples. In simulated gastrointestinal conditions, the SFD and FD cells show up to 4?h better tolerance than SD samples and unencapsulated cells in acidic and pepsin condition. The morphology of SFD samples shows particles almost in spherical shape with numerous fine pores, which in turn results in good rehydration behaviour of the powdered product.     

Hepatitis-B surface antigen (HBsAg) powder formulation: process and stability assessment

The purpose of this study was to develop a hepatitis-B surface antigen (HBsAg) dry powder vaccine formulation suitable for epidermal powder immunization (EPI) via an efficient, scalable powder-formation process. Several HBsAg dry powder formulations were prepared using four different powder-formation methods: freeze-drying/compress/grind/sieve (FD/C/G/S), spray-drying (SD), agarose beads, and spray freeze-drying (SFD). Powder properties and physical stability were determined using particle size analysis, tap density measurement, scanning electron microscopy, optical microscopy, and moisture content analysis. Physical, chemical and biochemical stability of HBsAg was determined by dynamic light scattering, an enzyme immune assay, and immunogenicity in a mouse or hairless guinea pig model. Out of the four powder-formation methods evaluated SFD outperformed other methods in the following considerations: good process efficiency, flexible scalability, and desirable particle characteristics for skin penetration. The stress posed by SFD appeared to be mild as HBsAg in the dry form retained its potency and immunogenicity. Notably, the mechanism of fast freezing by SFD actually promoted the preservation of HBsAg nanoparticle size, in good correlation with long-term biochemical stability. Among several formulations screened, the formulation containing 10 microg HBsAg in 1-mg powder with a tertiary mixture of trehalose, mannitol, and dextran, exhibited excellent overall stability performance. In conclusion, HBsAg dry powder formulations suitable for EPI were successfully prepared using SFD. Further, a systematic formulation development strategy allowed the development and optimization of an HBsAg dry powder formulation, demonstrating excellent long-term physical, biochemical, and immunological stability.     

Stabilization of alum-adjuvanted vaccine dry powder formulations: mechanism and application

Studies were performed to elucidate the mechanism of alum gel coagulation upon freezing and drying and its relationship to vaccine potency loss and to develop a novel freeze-drying process for the production of stable alum-adjuvanted vaccine formulations suitable for conventional needle injection and epidermal powder immunization (EPI). The alum hydroxide-adjuvanted hepatitis-B surface antigen (Alum-HBsAg) and the alum phosphate-adjuvanted diphtheria and tetanus toxoids (Alum-DT) were dehydrated by freeze drying (FD), spray drying (SD), air drying (AD), or spray freeze drying (SFD). After drying by FD, SD, or AD, alum gels coagulated when examined by optical microscopy and particle size analysis. In addition, desorption of antigen molecules from the coagulated when examined by optical microscopy and particle size analysis. In addition, desorption of antigen molecules from the coagulated alum gel upon reconstitution appeared to be difficult, as indicated by attenuated band intensity on SDS-PAGE. In contrast, SFD alum gels turned a homogenous suspension upon reconstitution, suggesting minimal alum coagulation. In the mouse model, the in vivo immunogenicity of SFD Alum-HBsAg was preserved, whereas the FD Alum-HBsAg suffered significant immunogenicity loss. Grinding of coagulated FD Alum-HBsAg into smaller particles could partially recover the immunogenicity. In a guinea pig study using EPI, the SD Alum-DT formulation was not immunogenic, but the SFD Alum-DT formulations had a vaccine potency comparable to that of the untreated DT administered by I.M. injection. Overall, the relationship of coagulation of alum gel upon reconstitution and the loss of vaccine potency was established in this study. Alum gels became highly coagulated after dehydration by spray drying and traditional freeze-drying processes. However, freezing rate played a critical role in preserving the adjuvant effect of alum and fast freezing decreased the tendency of alum coagulation. Spraying the alum gel into liquid nitrogen represents the fastest freezing rate achievable and resulted in no discernible alum coagulation. Therefore, SFD presents a novel and effective drying process for alum-adjuvanted vaccine formulations and is particularly valuable for dry powder applications such as EPI.     

A simple technique for hemoglobin estimation to screen for anaemia.

Drabkins method has been modified enabling detection of anaemia in a large population. 132 samples of EDTA blood were subjected to hemoglobin estimation by (1) Direct Drabkin (DD) (2), Filter Drabkin (FD) and (3) Special Filter Drabkin (SFD). Hemoglobin estimations by DD and FD compared well on statistical analysis. SFD with a punch diameter of 10.6 to 10.7 mm compared well with DD and is ideal for screening anaemia in field studies.     

Production of Inhalation Phage Powders Using Spray Freeze Drying and Spray Drying Techniques for Treatment of Respiratory Infections

Purpose

The potential of aerosol phage therapy for treating lung infections has been demonstrated in animal models and clinical studies. This work compared the performance of two dry powder formation techniques, spray freeze drying (SFD) and spray drying (SD), in producing inhalable phage powders.

Method

A Pseudomonas podoviridae phage, PEV2, was incorporated into multi-component formulation systems consisting of trehalose, mannitol and L-leucine (F1?=?60:20:20 and F2?=?40:40:20). The phage titer loss after the SFD and SD processes and in vitro aerosol performance of the produced powders were assessed.

Results

A significant titer loss (~2 log) was noted for droplet generation using an ultrasonic nozzle employed in the SFD method, but the conventional two-fluid nozzle used in the SD method was less destructive for the phage (~0.75 log loss). The phage were more vulnerable during the evaporative drying process (~0.75 log further loss) compared with the freeze drying step, which caused negligible phage loss. In vitro aerosol performance showed that the SFD powders (~80% phage recovery) provided better phage protection than the SD powders (~20% phage recovery) during the aerosolization process. Despite this, higher total lung doses were obtained for the SD formulations (SD-F1?=?13.1?±?1.7?×?10⁴ pfu and SD-F2?=?11.0?±?1.4?×?10⁴ pfu) than from their counterpart SFD formulations (SFD-F1?=?8.3?±?1.8?×?10⁴ pfu and SFD-F2?=?2.1?±?0.3?×?10⁴ pfu).

Conclusion

Overall, the SD method caused less phage reduction during the powder formation process and the resulted powders achieved better aerosol performance for PEV2.

     

A comparison between spray drying and spray freeze drying for dry powder inhaler formulation of drug-loaded lipid-polymer hybrid nanoparticles

Lipid-polymer hybrid nanoparticles - polymeric nanoparticles enveloped by lipid layers - have emerged as a potent therapeutic nano-carrier alternative to liposomes and polymeric nanoparticles. Herein we perform comparative studies of employing spray drying (SD) and spray freeze drying (SFD) to produce inhalable dry-powder form of drug-loaded lipid-polymer hybrid nanoparticles. Poly(lactic-co-glycolic acid), lecithin, and levofloxacin are employed as the polymer, lipid, and drug models, respectively. The hybrid nanoparticles are transformed into micro-scale nanoparticle aggregates (or nano-aggregates) via SD and SFD, where the effects of (1) different excipients (i.e. mannitol, polyvinyl alcohol (PVA), and leucine), and (2) nanoparticle to excipient ratio on nano-aggregate characteristics (e.g. size, flowability, aqueous reconstitution, aerosolization efficiency) are examined. In both methods, PVA is found more effective than mannitol for aqueous reconstitution, whereas hydrophobic leucineis needed to achieve effective aerosolization as it reduces nano-aggregate agglomeration. Using PVA, both methods are equally capable of producing nano-aggregates having size, density, flowability, yield and reconstitutibility in the range ideal for inhaled delivery. Nevertheless, nano-aggregates produced by SFD are superior to SD in terms of their aerosolization efficiency manifested in the higher emitted dose and fine particle fraction with lower mass median aerodynamic diameter.     

Formulation of pH responsive peptides as inhalable dry powders for pulmonary delivery of nucleic acids

Nucleic acids have the potential to be used as therapies or vaccines for many different types of disease, but delivery remains the most significant challenge to their clinical adoption. pH responsive peptides containing either histidine or derivatives of 2,3-diaminopropionic acid (Dap) can mediate effective DNA transfection in lung epithelial cells with the latter remaining effective even in the presence of lung surfactant containing bronchoalveolar lavage fluid (BALF), making this class of peptides attractive candidates for delivering nucleic acids to lung tissues. To further assess the suitability of pH responsive peptides for pulmonary delivery by inhalation, dry powder formulations of pH responsive peptides and plasmid DNA, with mannitol as carrier, were produced by either spray drying (SD) or spray freeze drying (SFD). The properties of the two types of powders were characterised and compared using scanning electron microscopy (SEM), next generation impactor (NGI), gel retardation and in vitro transfection via a twin stage impinger (TSI) following aerosolisation by a dry powder inhaler (Osmohaler™). Although the aerodynamic performance and transfection efficacy of both powders were good, the overall performance revealed SD powders to have a number of advantages over SFD powders and are the more effective formulation with potential for efficient nucleic acid delivery through inhalation.     

Comparative studies on physicochemical stability of cyclosporine A-loaded amorphous solid dispersions

The present study aimed to evaluate the physical stability on amorphous solid dispersion (SD) of cyclosporine A (CsA) employing hydroxypropyl cellulose (HPC). SD formulations (5-30% CsA) of CsA such wet-milled SD (WM/SD) and freeze-dried SD (FD/SD) were prepared, and both SD formulations were stored at 40 °C/75% relative humidity for 8 weeks. Transitions in morphology, dissolution behavior, crystallinity and thermal behavior of CsA were evaluated. There was at least 84-fold improvement in initial dissolution rate of SD formulations compared with that of amorphous CsA powder, although their dissolution rate was gradually decreased under accelerated conditions. In particular, aged FD/SD with a drug load of 30% exhibited highly limited dissolution as evidenced by 40% reduction of solubility after 8 weeks of storage. In contrast, aged WM/SD exhibited less reduction in dissolution rate compared with FD/SD. No significant changes were seen in crystallinity and thermal behavior after aging of SD formulations for 8 weeks; however, electron microscopic observations revealed aggregation of drug molecules/particles in the aged FD/SD, possibly leading to the reduced dissolution. From these findings, stability on CsA-loaded SD might be variable depending on the preparation methodology, and the wet-milling approach could be a viable option for preparing efficacious SD formulations with improved stability.     

Influenza vaccine powder formulation development: spray-freeze-drying and stability evaluation

The purpose of this study was to develop a spray-freeze-drying (SFD) process for preparing an influenza vaccine dry powder formulation suitable for epidermal powder immunization. After preformulation of two types of flu vaccines, their dry-powder formulations were prepared by SFD. Powder properties and physical stability were determined using particle size analysis, tap density measurement, scanning electron microscopy, optical microscopy, and moisture content analysis. Chemical and biochemical stability of vaccine antigens was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, single radial immunodiffusion assay, and in vivo immunogenicity in a mouse model. We demonstrated that SFD could produce high-density particles-a critical parameter for effective skin penetration. From the stability perspective, the stress posed by SFD was mild because the antigen in the dry powder retained its stability, potency, and immunogenicity. Among several formulations screened, we noted that formulation composition has a significant role in the powder's long-term physical and biochemical stability. One formulation, in particular, containing sub-unit vaccine (45 microg of antigen in 1 mg of powder) with a tertiary mixture of trehalose, mannitol, and dextran, exhibited excellent overall stability, including acceptable biochemical stability after being exposed to a highly humid environment. After all, we have not only demonstrated the suitability of SFD to prepare powders for epidermal powder immunization but also developed a systematic formulation development strategy that allowed the optimization of an influenza vaccine dry powder formulation. More important, this study led to the selection of a formulation system that had been successfully tested in a human clinical study.     

Spray freeze drying for dry powder inhalation of nanoparticles

Formulating nanoparticles for delivery to the deep lung is complex and many techniques fail in terms of nanoparticle stability. Spray freeze drying (SFD) is suggested here for the production of inhalable nanocomposite microcarriers (NCM). Different nanostructures were prepared and characterized including polymeric and lipid nanoparticles. Nanoparticle suspensions were co-sprayed with a suitable cryoprotectant into a cooled, stainless steel spray tower, followed by freeze drying to form a dry powder while equivalent compositions were spray dried (SD) as controls. SFD-NCM possess larger specific surface areas (67–77 m²/g) and lower densities (0.02 g/cm³) than their corresponding SD-NCM. With the exception of NCM of lipid based nanocarriers, SFD produced NCM with a mass median aerodynamic diameter (MMAD) of 3.0 ± 0.5 μm and fine particle fraction (FPF ⩽ 5.2 μm) of 45 ± 1.6% with aerodynamic performances similar to SD-NCM. However, SFD was superior to SD in terms of maintaining the particle size of all the investigated polymeric and lipid nanocarriers following reconstitution (S_f/S_i ratio for SFD ≈ 1 versus >1.5 for SD). The SFD into cooled air proved to be an efficient technique to prepare NCM for pulmonary delivery while maintaining the stability of the nanoparticles.     

Optimization of an Alum-Adsorbed Vaccine Powder Formulation for Epidermal Powder Immunization

Purpose. To develop stable and effective aluminum salt (alum)-adsorbed vaccine powder formulations for epidermal powder immunization (EPI) via a spray freeze-drying (SFD) process. Methods. Powder properties were determined using particle size analysis, tap density, and scanning electron microscopy. Alum coagulation was monitored via optical microscopy and particle sedimentation. Protein analysis was determined by the BCA protein assay, SDS-PAGE, and an enzyme immunoassay. In vivo immunogenicity and skin reactogenicity were performed on hairless guinea pigs and pigs, respectively. Results. SFD of hepatitis B surface antigen (HBsAg) adsorbed to aluminum hydroxide or aluminum phosphate using an excipient combination of trehalose/mannitol/dextran produced vaccine powders of dense particles and satisfactory powder flowability and hygroscopicity. This formulation also offered excellent long-term stability to the powder and the antigen. The two most important factors influencing alum particle coagulation are the freezing rate and the concentration of aluminum in the liquid formulation for SFD. The SFD vaccines, when delivered to hairless guinea pigs by EPI or injected intramuscularly after reconstitution, were as immunogenic as the original liquid vaccine. A further study showed that EPI with SFD alum-adsorbed diphtheria-tetanus toxoid vaccine was well tolerated, whereas needle injection of the liquid formulation caused persistent granuloma. Conclusions. Stabilization of alum-adsorbed vaccine by SFD has important implications in extending vaccination to areas lacking a cold chain for transportation and storage and may also accelerate the development of new immunization technologies such as EPI.     

维生素C乙基纤维素微囊的制备

以乙基纤维素为包囊材料,加入聚烯烃类包囊促进剂进行VC的包囊试验。微囊VC的溶出速率随包衣厚度递减。VC微囊片剂的体外溶出显著慢于普通片,放置后其变色亦较未经微囊处理者为缓。     

Preparation of DNA dry powder for non-viral gene delivery by spray-freeze drying: effect of protective agents (polyethyleneimine and sugars) on the stability of DNA

This study investigates the feasibility of using the process of spray-freeze drying (SFD) to produce DNA dry powders for non-viral gene delivery. The effect of protective agents was assessed on the stability of DNA dry powders after SFD. The process of SFD had adverse effects on the tertiary structure of DNA with the protective agents of sucrose, trehalose and mannitol. With the protection of these sugars, a band corresponding to the linear form of DNA was observed during gel electrophoresis between the supercoiled form (SC) and the open circular (OC) form. On the contrary, excess cationic condensing polyethyleneimine (PEI), in conjunction with the above sugars, had the ability to provide protection for DNA from degradation after SFD. This is indicated by the reservation in SC and OC forms of DNA during agarose gel electrophoresis. The electrostatic forces between PEI polymer and DNA are critical for providing protection against various stresses generated by the process of SFD. Furthermore, on rehydration, the particle size and zeta potential of PEI/DNA complexes at weight ratios 3:1 of SFD dry powders were well maintained. Also, no transfection activity loss of PEI/DNA complexes at weight ratios 3:1 on NIH/3T3 cells was observed for reconstituted powders as compared with untreated control solutions. These results give a better understanding of preparing stable DNA dry powders by the process of SFD.     

Spermined dextran, a cationized polymer, as absorption enhancer for pulmonary application of peptide drugs

Sperminated dextrans (SD) having different average molecular weights (MWs; 10, 40 and 70 kDa) and numbers of amino groups were prepared as cationized polymers for use as absorption enhancers. The absorption enhancing effects on the pulmonary absorption of insulin in rats and the permeation of FITC-dextran (MW 4,400, FD4) through calu-3 cell (human airway epithelial cell) monolayers by SD were evaluated. SD significantly enhanced the pulmonary absorption of insulin SD and the permeation of FD4 through calu-3 cells. The enhancing effects on the absorption insulin and permeation of FD4 through calu-3 cells increased with an increase in the molecular weigh of SD over the range 10-70 kDa. SD may interact directly with the luminal surface of mucus membranes via an ion-ion interaction and then induce signals that open tight junctions resulting in intercellular permeation of water soluble drugs. SD may be useful as an absorption enhancer for pulmonary delivery of peptide and protein drugs.     

Evaluation of the ulcerogenic effect of potassium chloride by endoscopy and fecal blood loss

In a blinded-observer crossover study, 16 volunteers ingested equimolar doses of three different potassium chloride preparations and placebo for nine days in a random order. The irritating effect on the mucosa was evaluated by means of upper gastrointestinal endoscopy and fecal blood loss, measured by reference to the amount of radioactivity in three 24-hour samples after previous injection of 51Cr-tagged autologous red cells. The microencapsulated KCl capsule preparation induced three ulcers, but the total endoscopic group score was not significantly higher than the score with placebo. The total endoscopic group score for the wax-matrix formulation was significantly higher than the score for placebo (P less than .05) and for a new microencapsulated KCl tablet preparation containing a potent disintegrant (P less than .10). We did not find any significant differences in mean fecal blood loss during the four different treatments. A correlation between endoscopic score and fecal blood loss was found in the placebo group (r = .54) and the microencapsulated KCl capsule preparation (r = .56) group.     

Stable high surface area lactate dehydrogenase particles produced by spray freezing into liquid nitrogen.

Enzyme activities were determined for lactate dehydrogenase (LDH) powder produced by lyophilization, and two fast freezing processes, spray freeze-drying (SFD) and spray freezing into liquid (SFL) nitrogen. The 0.25 mg/mL LDH aqueous feed solutions included either 30 or 100 mg/mL trehalose. The SFL process produced powders with very high enzyme activities upon reconstitution, similar to lyophilization. However, the specific surface area of 13 m(2)/g for SFL was an order of magnitude larger than for lyophilization. In SFD activities were reduced in the spraying step by the long exposure to the gas-liquid interface for 0.1-1s, versus only 2 ms in SFL. The ability to produce stable high surface area submicron particles of fragile proteins such as LDH by SFL is of practical interest in protein storage and in various applications in controlled release including encapsulation into bioerodible polymers. The SFL process has been scaled down for solution volumes <1 mL to facilitate studies of therapeutic proteins.     

海藻酸钠-聚左赖氨酸-海藻酸钠微囊包裹杂交瘤细胞的研究

目的对海藻酸钠-聚左赖氨酸-海藻酸钠(APA)微囊包裹杂交瘤细胞进行初步研究。方法用人IgG₁免疫小鼠，取免疫后脾细胞与小鼠骨髓瘤细胞系SP2/0细胞融合，获得分泌抗人IgGκ链单克隆抗体(mAb)的杂交瘤细胞株，命名为JY-A1；通过优化制备条件，制备包裹杂交瘤细胞的APA微囊，并考察形态学；比较不同微囊膜的机械强度和化学强度；用ELISA法测定mAb的释放。小鼠腹腔注射微囊，不同时间回收观察。结果相同条件下制得的微囊粒径均匀、表面光滑。体外培养条件下mAb能持续透过微囊膜。小鼠腹腔注射微囊后无异常，回收的微囊大部分形态完整。结论采用高压静电成囊技术制备的APA微囊具有较高的膜强度，对细胞分泌产物mAb能持续稳定释放，在小鼠体内有较好的生物相容性。     

Microencapsulation of menadione sodium bisulphite with polydimethylsiloxane by the spray-drying process: characterization by thermal analysis.

Menadione sodium bisulphite was microencapsulated with a polydimethylsiloxane membrane using spray-drying technology. Tests were performed using laboratory equipment and a Niro Atomizer pilot plant to scale up the process. The products were characterized with differential thermal analysis (DTA) and chemical and physical methods. Many differences between raw material and microencapsulated powder result from DTA data. The thermal characterization confirms that the spray-drying microcoating could be used to protect powder from the oxidative actions of the atmosphere.     

Prolongation of the effective duration of cytomedical therapy by re-injecting SK2 hybridoma cells microencapsulated within alginate-poly(L)lysine-alginate membranes into human interleukin-6 transgenic mice

We previously reported that SK2 hybridoma cells that secreted anti-human interleukin-6 (hIL-6) monoclonal antibodies (SK2 mAb) were microencapsulated within alginate-poly(L)lysine-alginate (APA) membranes (APA-SK2 cells) for immunoisolation, and a single intraperitoneal injection of these APA-SK2 cells remarkably improved IgG1 plasmacytosis in hIL-6 transgenic mice (hIL-6 Tgm). However, the duration of the effectiveness of APA-SK2 cells as a cytomedicine was unfortunately limited. In this study, we attempted to re-inject APA-SK2 cells into hIL-6 Tgm for the purpose of prolonging the cytomedical therapy. In hIL-6 Tgm re-injected with APA-SK2 cells, the plasma IgG1 level did not show any increase in 37 week old mice, and their survival time was at least three times longer than those of untreated hIL-6 Tgm. These results suggest that re-injected APA-SK2 cells survived and secreted SK2 mAb in the allogeneic mice. Thus, the limited duration of the cytomedical effects of APA-SK2 cells was probably caused by the disappearance of the inner space of microcapsules for cell proliferation, not by the rejection of the host's immune system. Therefore, if we can regulate the proliferation of the cells microencapsulated within a semipermeable membrane, we may be able to develop a cytomedicine which will continue its function longer after a single injection.     

A novel apparatus for rat in vivo evaluation of dry powder formulations for pulmonary administration

The lungs have attracted increasing attention as a site for administration of drugs, including macromolecules that are poorly absorbed from the intestine. There have been a number of basic studies in which peptide solutions were administered to experimental animals via the lungs. Although there have been several studies of pulmonary peptide absorption from dry powder formulations, a simpler and more inexpensive apparatus for administration of dry powders would enhance rapid screening of the formulations. In this study, we developed a simple apparatus to disperse dry powders. The apparatus has two 3-way stopcocks; one allows dispersal of powders at a constant pressure and airflow, and the other allows rats to breathe before and after administration. Dry powders of fluorescein (FL) and FITC-dextran (FD4) were manufactured by the spray-drying technique. The effects of operating conditions on the absorption of these model drugs were examined in rats. The C(max) for FL from dry powder was lower than that from solution and mean residence time was extended, suggesting that dissolution was the rate-determining step for FL absorption from dry powder. For FD4, the rate of absorption may not be regulated by dissolution but by epithelial transport. Absorption of insulin from spray-dried powder via the rat trachea was investigated using this apparatus. Intratracheally administered spray-dried insulin powder decreased plasma glucose level to a greater extent than spray-dried insulin solution administered via the same route. Thus, the apparatus is simple, inexpensive, and useful for rapid screening of dry powder formulations.