Protein/peptide drugs are living, active molecules made from amino acids that easily bind to receptors and have little side effects. But protein/peptide drugs have small molecular weights, poor gut penetration and stability, and very short half-lives due to hydrolysis, enzymatic hydrolysis and the surface charge of the drug. So, most of them demand a lot of injections and that's putting the patient at increased risk. So scientists have been obsessed with the effects of protein/peptide agents over the long-term. Of these, switching dose forms during long-acting drug delivery can both lower costs and accelerate the time to market for a drug – a promising pathway for long-acting studies.
Figure 1. A diagram of PLGA-based biodegradable microspheres in drug delivery. (Su Y, et al.; 2021)
Microspheres can achieve targeted and oral delivery of protein/peptide drugs, and through the selection of carrier materials and preparation processes, they can also control the release duration and release behavior of the drug, so they are widely used. Microsphere products prepared using biodegradable materials as carriers can be released in the body for several months, significantly reducing the frequency of administration and improving patient compliance. However, the main obstacle in the development of protein/peptide drug microsphere preparations is that due to the instability, hydrophilicity and large size of protein/peptide drugs, their microsphere preparations are prone to burst release, delayed release, and incomplete release, and loss of activity, leading to fluctuations in blood drug concentration, thereby indirectly or directly causing adverse reactions. This review summarizes the influencing factors on the development of protein/peptide drug microsphere preparations, and reviews various improvement strategies in terms of carrier materials and preparation processes, with a view to providing new ideas for the development of protein/peptide drug microsphere products.
Existing Obstacles
Drug dispersal from poly(lactide-co-glycolide) (PLGA) microspheres is influenced by various processes like PLGA water retention, hydrolysis, erosion and drug-water diffusion. So the drug release from the microsphere is generally a burst release, a delayed release, and a rapid release. As well as the above, burst release of drugs is also dependent on the microsphere particle size, relative molecular weight of polymers, porosity, drug composition and drying process. Burst release not only affects blood drug levels but high levels of a drug released can over-do it, which can make blood drug levels out of the therapeutic range, with grave side effects. Moreover, if you give too much drug during the initial phase, you will also decrease the overall time for effective dose.
Hysteresis refers to the phenomenon that drugs are slowly released or not released from a preparation, which usually occurs sometime after the burst release or initial release. It is speculated that the reason for the hysteresis is that the polymer chains are excessively entangled, forming a glassy state, which exhibits the characteristics of high fluidity and low water absorption, resulting in a reduction in diffusion channels in the polymer microspheres and difficulty in drug release. The higher the relative molecular mass of PLGA and the stronger its hydrophobicity, the slower the rate at which the solution penetrates into the polymer, so the lag phase will be longer. At this time, the lag phase can be shortened by increasing the pores in the microspheres.
Incomplete release of protein/peptide drugs in microspheres is common and it has more to do with degradation, conformational modification and aggregate protein/peptide drugs and adsorption of carrier materials.
- Degradation, Conformational Change and Aggregation of Protein/Peptide Drugs
The stability of protein/peptide drugs is easily affected by the external environment. Shear stress, interfacial stress, dehydration stress, etc. during the preparation process will cause their degradation and affect their activity. When protein/peptide drugs are exposed to the oil/water or water/air interface, or come into contact with cross-linking agents, their conformation may change and spontaneously aggregate. It is reported that peptides and proteins with antiparallel β-folded fragments between molecules are prone to aggregate at the oil/water interface, which leads to reduced drug efficacy and antibody response. For example, erythropoietin used to treat anemia is prone to form aggregates that cause anemia during the microspherization process, and the therapeutic drug is transformed into a pathogenic factor. In order to inhibit aggregation, stabilizers or more hydrophilic oil phases can be used in the preparation of microspheres, and water-in-oil-in-solid (S/O/W) emulsification methods can be used.
- Adsorption and Acylation with Carrier Materials
Proteins/peptides can bind to degraded or undegraded polymers through nonspecific adsorption and ionic interactions, resulting in incomplete drug release. Peptides can undergo acylation side reactions with PLGA or its degradation products to form peptide-PLGA peptidyl adducts. It has been reported that the addition of divalent cations such as Ca2+ and Mn2+ to PLGA can competitively inhibit the adsorption reaction of peptides to PLGA.
Modification of Carrier Materials
PLGA has been approved by the US FDA for the preparation of protein/peptide drug microspheres due to its excellent mechanical strength and biocompatibility, but its inherent hydrophobicity is not conducive to interaction with cells. In addition, lactic acid produced by PLGA degradation can also induce inflammatory responses and affect the stability of protein/peptide drugs. Therefore, improving the hydrophobicity of PLGA through modification can improve drug delivery efficiency.
- Copolymerization Modification
PLGA will transform in form and structure after polymerization with other polymers that could have different drug release requirements. But the metabolic circuitry of this kind of polymer carrier and the mechanism by which the carrier interacts with the drug remain to be discovered. The hydrophilic components like PEG and poloxamer, for instance, can be copolymers of PLGA with linear or nonlinear multi-block copolymers such as diblock and triblock. Having more polar segments helps the copolymer become more hydrophilic, which is good for keeping protein/peptide drugs stable. PLGA can also be copolymerised with hydrophobic components like polycaprolactone (PCL). The addition of caprolactone units can decrease the extent to which the polymer backbone is subject to acidic degradation products. Compared to PLGA microspheres, copolymer microspheres containing caprolactone units are longer release time with variable drug release rate.
Compared with forming new copolymers through copolymerization, it is much easier to prepare microspheres by mixing PLGA with other materials. Hydrophilic materials such as cyclodextrin, chitosan, and polyethyleneimine can be blended with PLGA as exogenous porogens to regulate the release of microspheres. In addition, microspheres can also be prepared by mixing PLGAs of different relative molecular weights and specifications. Low molecular weight PLGA will degrade rapidly during the release phase, and the acidic degradation products produced and accumulated can act as endogenous porogens to accelerate the degradation of high molecular weight PLGA.
Improvement of Preparation Technology
The emulsified solvent evaporation method is a common method for preparing protein/peptide drug microspheres. Protein/peptide drugs can be dissolved in aqueous solution as the inner aqueous phase (W1), or in the form of solid powder as the inner solid phase (S); W1 or S is then homogenized with the oil phase containing PLGA to form colostrum; the resulting colostrum is injected into the outer aqueous phase under high-speed shear to form a W1/O/W2 or S/O/W type emulsion, and the organic solvent is finally evaporated and solidified to obtain microspheres. However, during the preparation process, proteins/peptides are easily affected by the oil/water interface and shear force, so a strategy of pre-encapsulating proteins/peptides can be considered.
It has been reported that affinity hydrogels can release protein drugs at a constant rate. In the process of microsphere preparation, the use of drug-loaded thermosensitive gel (below the gelation temperature, the thermosensitive gel encapsulates the drug in the form of micelles; above the gelation temperature, the molecular chains entangle with each other to form a gel) as the inner water phase can not only avoid the destruction of protein/peptide drugs by harsh environments such as organic solvents and severe shear, but also increase the viscosity of the inner water phase to prevent drug leakage.
- Inner Aqueous Phase Liposomes
Liposomes are highly biocompatible and surface customizable, but their membrane can be ruptured. Others have packed liposomes into hard PLGA microspheres to form a novel drug delivery system liposome microsphere (LIM), which adds the benefits of the two and has potential to protect the biological function of protein/peptide drugs and target delivery.Inner Aqueous Phase Micelles
Micelles will also self-assemble in the solution to a core-shell, and can be stabilised by encapsulating proteins inside micelles. Some researchers developed a biodegradable bile acid-PLGA-b-(polyethyleneimine-polyethylene glycol) [CAPLGA-b-(PEI-PEG)] copolymer that can self-assemble into cationic micelles and load insulin by electrostatic means. We employed the insulin micelles as the inner water phase to form the microspheres by W1/O/W2 emulsion process. Compared to insulin microspheres devoid of micelles, insulin micelle microspheres release less burst, have greater bioavailability and longer duration hypoglycemic effect, while the micelles' proton-buffering ability can shield insulin from the acidic microenvironment that arises from PLGA degradation.
- Internal Solid Phase Solid Dispersion
In solid dispersions, drugs can exist in amorphous, molecular or microcrystalline forms and remain highly dispersed.
- Internal Solid Phase Lyophilized Complex
Protein/peptide drugs can also be mixed with other molecules and lyophilized to form a complex. It is reported that dissolving GOS with poloxamer or PEG and freeze-drying them, and then using the lyophilized complex as the internal solid phase to prepare microspheres can greatly improve the encapsulation rate and bioavailability of GOS.
The rapid solidification of microspheres helps to improve the encapsulation rate of drugs, shorten the contact between protein/peptide drugs and the oil/water interface, and reduce the loss of drug activity. Therefore, it is recommended to use highly volatile organic solvents. DCM is volatile and has good solubility for PLGA. It is widely used to prepare microspheres; but DCM is carcinogenic, and the residual amount should be strictly controlled during preparation. Ethyl acetate has low toxicity and is considered to be a substitute for DCM; but ethyl acetate has a weak solubility for PLGA and may form PLGA fibrous aggregates, resulting in a decrease in encapsulation rate.
- Modification of the External Aqueous Phase
Typically, the greater the osmotic pressure of the external aqueous phase, the less water will diffuse to the microspheres and simultaneously, the less drugs will diffuse to the external aqueous phase, the less pores are generated in the microspheres, and the higher the encapsulation rate and porous surface. Protein/peptide drugs are not solubilized at the isoelectric point, and the pH of the outer aqueous phase can be regulated based on this characteristic.
New Preparation Technology
The particle size of microspheres prepared by emulsification solvent evaporation and spray drying is uneven and needs further screening, while microfluidic technology can achieve precise control of multiple fluids to obtain microspheres with uniform particle size, small coefficient of variation and adjustable structure. In large-scale production, microfluidic technology has good reproducibility, but the production efficiency is difficult to meet the needs, and it is currently mainly used in laboratory research. The microspheres obtained by microfluidic technology have uniform particle size and concentrated release, which can be used for pulse administration.
- Supercritical Fluid (SCF) Technology
Preparing microspheres usually involves a lengthy phase change in order to expel organic solvents, and the slower solvent expulsion may release the drug into the outward aqueous phase, where it will be clinging to the microspheres' surface leading to burst release and slow encapsulation rate. SCF technology has high mass transfer capacity, rapid removal of organic solvents, light processing, does not need to be heated and contains low residual organic solvents, thus can mitigate the above-mentioned issues.
- Self-healing Encapsulation Technology
Self-healing encapsulation refers to mixing pre-prepared blank porous microspheres with drug solutions, allowing the drugs to spontaneously diffuse into the microspheres, and then raising the temperature to above the glass transition temperature of the polymer or using a blocking solvent to close the pores of the microspheres. Self-healing encapsulation can reduce the effects of shear stress and exposure to the oil/water interface on protein/peptide drugs, and the microspheres can be sterilized before loading the drugs to avoid the loss of activity of protein/peptide drugs due to exposure to the sterilization process. Therefore, self-healing encapsulation technology is very friendly to sensitive biomolecules, and the preparation process is relatively simple. The only drawback is the low loading capacity. However, by adjusting the process parameters such as microsphere porosity, open/closed pores, drug solution concentration, and sealing conditions, a better encapsulation effect can also be achieved. Studies have shown that blank wet microspheres have a better loading capacity. Compared with the loading capacity of 15.1µg/mg of dry microspheres, wet microspheres can reach 68µg/mg.
References
- Su Y, et al.; PLGA-based biodegradable microspheres in drug delivery: recent advances in research and application. Drug Deliv. 2021, 28(1):1397-1418.
- Rahmani F, et al.; The recent insight in the release of anticancer drug loaded into PLGA microspheres. Med Oncol. 2023, 40(8):229.
