Nanoprecipitation versus emulsion-based techniques for the encapsulation of proteins into biodegradable nanoparticles and process-related stability issues

The goal of this study was to investigate the entrapment of 3 different model proteins (tetanus toxoid, lysozyme, and insulin) into poly(D,L-lactic acid) and poly(D,L-lactic-co-glycolic acid) nanoparticles and to address process-related stability issues. For that purpose, a modified nanoprecipitation method as well as 2 emulsion-based encapsulation techniques (ie, a solid-in oil-in water (s/o/w) and a double emulsion (w1/o/w2) method) were used. The main modification of nanoprecipitation involved the use of a wide range of miscible organic solvents such as dimethylsulfoxide and ethanol instead of the common acetone and water. The results obtained showed that tetanus toxoid and lysozyme were efficiently incorporated by the double emulsion procedure when ethyl acetate was used as solvent (>80% entrapment efficiency), whereas it was necessary to use methylene chloride to achieve high insulin entrapment efficiencies. The use of the s/o/w method or the formation of a more hydrophobic protein-surfactant ion pair did not improve protein loading. The nanoprecipitation method led to a homogenous population of small nanoparticles (with size ranging from ≈130 to 560 nm) and in some cases also improved experimental drug loadings, especially for lysozyme (entrapment efficiency >90%). With respect to drug content determination, a simple and quick matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method provided results very close to those obtained by reverse phase-high-performance liquid chromatography. With respect to protein stability, the duration and intensity of sonication were not a concern for tetanus toxoid, which retained more than 95% of its antigenicity after treatment for 1 minute. Only a high methylene chloride:water ratio was shown to slightly decrease toxoid antigenicity. Finally, no more than 3.3% of A21 desamido insulin and only traces of covalent insulin dimer were detected in nanoparticles. In conclusion, both the double emulsion and nanoprecipitation methods allowed efficient protein encapsulation. MALDI-TOF MS allowed accurate drug content determination. The manufacturing processes evaluated did not damage the primary structure of insuli

Polymer-based nanoparticles loaded with a TLR7 ligand to target the lymph node for immunostimulation

Small-molecule agonists for the Toll-like receptors (TLR) 7 and 8 are effective for the immunotherapy of skin cancer when used as topical agents. Their systemic use has however been largely unsuccessful due to dose-limiting toxicity. We propose a polymer-based nanodelivery system to target resiquimod, a TLR7 ligand, to the lymph node in order to focus the immunostimulatory activity and to prevent a generalized inflammatory response. We demonstrate successful encapsulation of resiquimod in methoxypoly(ethylene glycol)-b-poly(DL-lactic acid) (mPEG-PLA) and mixed poly(DL- lactic-co-glycolic acid) (PLGA)/mPEG-PLA nanoparticles. We show that these particles are taken up mainly by dendritic cells and macrophages, which are the prime initiators of anticancer immune responses. Nanoparticles loaded with resiquimod activate these cells, demonstrating the availability of the immune-stimulating cargo. The unloaded particles are non-inflammatory and do not have cytotoxic activity on immune cells. Following subcutaneous injection in mice, mPEG-PLA and PLGA/mPEG-PLA nanoparticles are detected in dendritic cells and macrophages in the draining lymph nodes, demonstrating the targeting potential of these particles. Thus, polymer-based nanoparticles represent a promising delivery system that allows lymph node targeting for small-molecule TLR7 agonists in the context of systemic cancer immunotherapy

Development of resiquimod-loaded modified PLA-based nanoparticles for cancer immunotherapy: A kinetic study

Resiquimod (R848), a member of the imidazoquinoline family, is a Toll-like receptor 7/8 agonist with high potency for cancer immunotherapy. However, tolerance induction and adverse effects limit its development as a drug. Encapsulation in a polymer matrix can circumvent these limitations, as shown in our formerly published approach where R848 was loaded into polylactic acid (PLA)-based nanoparticles (NP). Although the results were encouraging, low rates of encapsulation and rapid release of the drug were observed. In this study, we present a new strategy using mixed NP from modified linear PLA in order to improve the encapsulation and modulate the release profile of R848. Modified PLA polymers were designed and synthesized by microwave-assisted ring opening polymerization of d,l-lactide, using polyethylene glycol as initiator to increase the hydrophilic properties of the polymer or linear saturated aliphatic chains (C8 or C20) to increase the affinity with hydrophobic R848. NP were prepared by solvent evaporation method, leading to particles of 205–288 nm loaded with either R848 or DiO as a tracking agent. The release profile showed longer retention of R848 at both neutral and acidic pH for NP from grafted polymers. Upon exposure to phagocytic immune cells, NP were actively taken up by the cells and no impact on cell viability was observed, independently of the constitutive polymer. All R848-loaded NP activated macrophages to secrete interleukin-6, demonstrating that the drug cargo was immunologically active. Importantly, macrophage activation by NP- delivered R848 was slower than with free R848, in accordance with the in vitro release profiles. Thus, NP prepared from modified PLA polymers showed no signs of toxicity to immune cells and efficiently delivered their immunoactive cargo in a delayed manner. This delivery strategy may enhance the efficacy and safety of small-molecule immunostimulants

Protein Drugs Entrapped Within Micro- & Nanoparticles: An Overview of Therapeutic Challenges & Scientific Issues

The ever-growing market of protein-based therapeutics as well as the advent of biodegradable and non-toxic polymers has boosted research in the field of protein encapsulation using nano- and microparticles. Although such small carriers are promising tools for protein administration, hurdles still exist, making protein formulation a difficult task. This review discusses the scientific issues that should be taken into account when encapsulating therapeutic protein drugs into biodegradable polymeric nano- and microparticles. It mostly deals with the manufacturing procedures of the particles and with the analysis of the encapsulated proteins. Sample preparation prior to qualitative or quantitative analysis is discussed together with the analytical methods employed. Indeed, the entrapped protein can be correctly analyzed only if the sample preparation and drug analysis minimize drug loss and degradation. The most common encountered pitfalls are reviewed and commented upon to emphasize specific concerns, which require closer scrutiny.</p

Development of a nanoprecipitation method intended for the entrapment of hydrophilic drugs into nanoparticles

This study investigates formulation and process modifications to improve the versatility of the nanoprecipitation technique, particularly with respect to the encapsulation of hydrophilic drugs (e.g. proteins). More specifically, the principal objective was to explore the influence of such modifications on nanoparticle size. Selected parameters of the nanoprecipitation method, such as the solvent and the non-solvent nature, the solvent/non-solvent volume ratio and the polymer concentration, were varied so as to obtain polymeric nano-carriers. The feasibility of such a modified method was assessed and resulting unloaded nanoparticles were characterized with respect to their size and shape. It was shown that the mean particle size was closely dependent on the type of non-solvent selected. When alcohols were used, the final mean size increased in the sequence: methanol < ethanol < propanol. Surfactants added to the dispersing medium were usually unnecessary for final suspension stabilization. Changing the solvent/non-solvent volume ratio was also not a determinant factor for nanoparticle formation and their final characteristics, provided that the final mixture itself did not become a solvent for the polymer. A too high polymer concentration in the solvent, however, prevented nanoparticle formation. Both poly(lactic acid) (PLA) and poly(d,l-lactic-co-glycolic acid) (PLGA) could be used by accurately choosing the polymer solvent and in this respect, some non-toxic solvents with different dielectric constants were selected. The nanoparticles obtained ranged from about 85–560 nm in size. The nanoparticle recovery step however needs further improvements, since bridges between particles which cause flocculation could be observed. Finally, the presented results demonstrate that the nanoprecipitation technique is more versatile and flexible than previously thought and that a wide range of parameters can be modified

Sonification parameters for the preparation of biodegradable nanocapsules of controlled size by the double emulsion method

The main goal of the present work was to study the influence of the sonication process on the characteristics of poly(lactide-co-glycolide) nanocapsules prepared by the water-in-oil-in-water solvent evaporation method. The duration and intensity of sonication were investigated with respect to their ability to modify the size and distribution of the nanocapsule population. It has been demonstrated that the duration of the second mixing step (leading to the w/o/w emulsion) is of greater influence than that of the first step (water-in-oil emulsion) on the final mean particle size. A three-dimensional response surface was drawn up to show that when the second emulsification time increased, the mean size decreased until reaching a plateau. A threshold in sonication intensity also exists, allowing optimization of both parameters and the formation of nanocapsules of controlled size with a rather narrow distribution. The use of a vortex mixer instead of a sonicator during the first mixing step led to nanocapsules with a similar response surface, supporting the idea that the second step is the decisive one. Finally, nanocapsules loaded with methylene blue, a hydrophilic model compound with a positive charge, were characterized and the encapsulation efficiency calculated. Their size and distribution were similar to that of the blank nanocapsules. Entrapment efficiency was independent of duration, intensity, and type of mixing. From these results, it was concluded that nanocapsules of controlled size could be obtained upon optimizing certain process parameters

Strategic approaches for overcoming peptide and protein instability within biodegradable nano- and microparticles

This paper reviews the major factors that are closely involved in peptide and protein degradation during the preparation of biodegradable nano- and microparticles. The various means usually employed for overcoming these obstacles are described, in order to bring to the fore the strategies for protein stabilization. Both processing and formulation parameters can be modified and are distinctly considered from a strategic point of view. We describe how partial or full protein stability retention within the carriers and during drug release might be achieved by individual or combined optimized strategies. Additionally, problems commonly encountered during protein quantification, stability determination and release are briefly reviewed. Artefacts that might occur during sampling and analytical procedures and which might hinder critical interpretation of results are discussed