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Minimizing acylation of peptides in PLGA microspheres.

ABSTRACT: The main objective of this study was to characterize and find mechanisms to prevent acylation of therapeutic peptides encapsulated in glucose-star poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres. The effect of addition of divalent cation salts CaCl(2), MnCl(2) as well as carboxymethyl chitosan (CMCS) on inhibition of acylation of octreotide (Oct), salmon calcitonin (sCT), and human parathyroid hormone (hPTH) was evaluated. Peptide content and integrity inside the degrading microspheres was monitored by reversed-phase high performance liquid chromatography (HPLC) and mass spectrometry during release incubation under physiological conditions. The extent of peptide acylation was strongly inhibited in the formulations containing divalent cations and/or CMCS as excipients, although specific effects were dependent on the specific peptide and excipient combinations. Both inorganic cations improved stability of Oct and hPTH but not sCT. Addition of CMCS alone was ineffective. Combining inorganic cations with CMCS improved stability of Oct and sCT but it had no effect on hPTH stability. The operative stabilization mechanisms are consistent with blocking peptide-PLGA interactions by a) directly competing for PLGA interactions with dications and/or b) increasing peptide affinity in the stabilizer phase within PLGA pores. Hence, inorganic multivalent cations are general stabilizers against peptide acylation, the effect of which may be augmented in certain instances with addition of CMCS.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC4261194 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Minimizing acylation of peptides in PLGA microspheres.

Zhang Ying Y Schwendeman Steven P SP

Journal of controlled release : official journal of the Controlled Release Society 20120421 1

The main objective of this study was to characterize and find mechanisms to prevent acylation of therapeutic peptides encapsulated in glucose-star poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres. The effect of addition of divalent cation salts CaCl(2), MnCl(2) as well as carboxymethyl chitosan (CMCS) on inhibition of acylation of octreotide (Oct), salmon calcitonin (sCT), and human parathyroid hormone (hPTH) was evaluated. Peptide content and integrity inside the degrading microspheres was ...[more]

PMID: 22546683

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Project description:Herein, we describe the detailed development of a simple and effective method to microencapsulate vaccine antigens in poly(lactic-co-glycolic acid) (PLGA) by simple mixing of preformed active self-microencapsulating (SM) PLGA microspheres in a low concentration aqueous antigen solution at modest temperature (10-38 °C). Co-encapsulating protein-sorbing vaccine adjuvants and polymer plasticizers were used to "actively" load the protein in the polymer pores and facilitate polymer self-healing at a temperature>the hydrated polymer glass transition temperature, respectively. The microsphere formulation parameters and loading conditions to provide optimal active self-healing microencapsulation of vaccine antigens in PLGA was investigated. Active self-healing encapsulation of two antigens, ovalbumin and tetanus toxoid (TT), in PLGA microspheres was adjusted by preparing blank microspheres containing different vaccine adjuvants (aluminum hydroxide (Al(OH)₃) or calcium phosphate). Active loading of vaccine antigen in Al(OH)₃-PLGA microspheres was found to: a) increase with an increasing loading of Al(OH)₃ (0.88-3 wt.%) and addition of porosigen, b) decrease when the inner Al(OH)₃/trehalose phase to 1 mL outer oil phase and size of microspheres was respectively >0.2 mL and 63 μm, and c) change negligibly by PLGA concentration and initial incubation (loading) temperature. Encapsulation of protein sorbing Al(OH)₃ in PLGA microspheres resulted in suppression of self-healing of PLGA pores, which was then overcome by improving polymer chain mobility, which in turn was accomplished by coincorporating hydrophobic plasticizers in PLGA. Active self-healing microencapsulation of manufacturing process-labile TT in PLGA was found to: a) obviate micronization- and organic solvent-induced TT degradation, b) improve antigen loading (1.4-1.8 wt.% TT) and encapsulation efficiency (~97%), c) provide nearly homogeneous distribution and stabilization of antigen in polymer, and d) provide improved in vitro controlled release of antigenic TT.

Project description:Vaccines are a critical clinical tool in preventing illness and death due to infectious diseases and are regularly administered to children and adults across the globe. In order to obtain full protection from many vaccines, an individual needs to receive multiple doses over the course of months. However, vaccine administration in developing countries is limited by the difficulty in consistently delivering a second or third dose, and some vaccines, including the inactivated polio vaccine (IPV), must be injected more than once for efficacy. In addition, IPV does not remain stable over time at elevated temperatures, such as those it would encounter over time in the body if it were to be injected as a single-administration vaccine. In this manuscript, we describe microspheres composed of poly(lactic-co-glycolic acid) (PLGA) that can encapsulate IPV along with stabilizing excipients and release immunogenic IPV over the course of several weeks. Additionally, pH-sensitive, cationic dopants such as Eudragit E polymer caused clinically relevant amounts of stable IPV release upon degradation of the PLGA matrix. Specifically, IPV was released in two separate bursts, mimicking the delivery of two boluses approximately one month apart. In one of our top formulations, 1.4, 1.1, and 1.2 doses of the IPV serotype 1, 2, and 3, respectively, were released within the first few days from 50mg of particles. During the delayed, second burst, 0.5, 0.8, and 0.6 doses of each serotype, respectively, were released; thus, 50mg of these particles released approximately two clinical doses spaced a month apart. Immunization of rats with the leading microsphere formulation showed more robust and long-lasting humoral immune response compared to a single bolus injection and was statistically non-inferior from two bolus injections spaced 1 month apart. By minimizing the number of administrations of a vaccine, such as IPV, this technology can serve as a tool to aid in the eradication of polio and other infectious diseases for the improvement of global health.

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Minimizing acylation of peptides in PLGA microspheres.

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Minimizing acylation of peptides in PLGA microspheres.

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