Microencapsulation of protein‐loaded polysaccharide particles within poly(D,L‐lactic‐co‐glycolic acid) microspheres using S/O/W: characterization and release studies

This report demonstrates a process to form polysaccharide glassy particles without water–oil or water–air interfaces as well as ionic polymers and its application in formulating sustained‐release dosage forms for structurally delicate proteins. When a co‐solution containing dextran and polyethylene glycol (PEG) was subjected to freeze‐drying, the dextran separated out of the solution to form dispersed phases surrounded by a PEG‐rich continuous phase and was solidified during subsequent lyophilization to fine glassy particles, 1–2 µm in diameter. Water‐soluble proteins can easily be loaded in these glassy particles due to preferential partition and become resistant to organic solvents simply by adding them into the dextran‐PEG co‐solution. After washing away the PEG continuous phase with organic solvents, the protein‐containing glassy particles can be suspended in a hydrophobic polymer solution and formulated into various pharmaceutical dosage forms and medical devices for sustained‐release protein delivery. In the present study, myoglobin, bovine serum albumin (BSA), and β‐galactosidase (β‐gal) were formulated in PLGA mcirospheres and as model proteins using this glassy particulate approach, and subjected to a series of assays for release kinetics, structural integrity, and bioactivity. The experimental results indicated that this system offered well preserved protein integrity and bioactivity as well as significantly improved protein release kinetics. Copyright © 2008 John Wiley & Sons, Ltd.     

Incorporation of water-soluble drugs in PLGA microspheres

Poly(lactide-co-glycolide) (PLGA) microspheres containing blue dextran, as a model of water-soluble drugs, were prepared from w₁/o/w₂ emulsions by using a microhomogenizer and a solvent evaporation method. Effects of preparation conditions, such as, concentration of poly(vinyl alcohol) (PVA) in w₂ phase, viscosity of inner soluble water phase, volume ratio of oil phase to w₁ phase in primary emulsion, PLGA concentration in oil phase, and molecular weight or composition of PLGA, upon the properties of PLGA microspheres containing water-soluble drugs were examined. Concentration of poly(vinyl alcohol) (PVA), the dispersant dissolved in w₂ phase of secondary emulsion did not show any effects on the final particle size. On the other hand, volume ratio of oil phase to water one in primary emulsion affected the final particle size, which seemed to be related to the local PLGA concentration in w₁/o emulsions. That is, the particle size increased as the volume ratio of w₁ phase against oil phase, w₁/o (v/v), increased. The loading efficiency, however, was not affected by the volume ratio of w₁/o (v/v), but affected by blue dextran concentration in w₁ phase. Higher loading efficiency was observed in PLGA microspheres prepared from w₁ phase containing lower concentration of blue dextran. Blue dextran solution (inner water phase) with the lower viscosity may result in the lower leakage ratio of blue dextran during the preparation procedure. Increases in concentration and molecular weight of PLGA made particle size larger.     

Enhanced gentamicin loading and release of PLGA and PLHMGA microspheres by varying the formulation parameters

The purpose of this study was to develop a suitable formulation for gentamicin sulfate (GS) that gives a sustained release of the drug. Therefore this drug was loaded into poly(D,L-lactide-co-glycolide) (PLGA) and poly(lactic-co-hydroxymethyl glycolic acid) (PLHMGA) microspheres. The effects of various formulation parameters (ethanol, surfactant, osmotic value of the external phase, polymer type and concentration) on particle characteristics (size, loading and release) were investigated. The GS loaded microspheres were prepared using a double emulsion evaporation technique. The results demonstrate that neither ethanol nor surfactants had beneficial effects on the drug loading efficiency (around 4-10%). However, an increase in buffer concentration (and thus osmotic pressure) of the external phase resulted in a substantial increase of GS-loading (from 10 to 28%). Further, an increase of concentration of PLGA in DCM from 10% to 15/20% caused a 4-time increase of the drug loading. The best formulation identified in this study had a loading efficiency of around 70% resulting in PLGA microspheres with a 6% (w/w) loading. The particles showed a burst release of the drug depending on their porosity, followed by a phase of 35 days where hardly any release occurred. The drug was then slowly released for around 25 days likely due to degradation of the microspheres. The drug loading efficiency of GS in PLHMGA was not significantly different from PLGA microspheres (64%). The release of GS from PLHMGA microspheres was faster than that of PLGA because the degradation rate of PLHMGA is more rapid than PLGA. This study shows that prolonged release of gentamicin can be obtained by loading this drug into microspheres made of biodegradable aliphatic polyesters.     

Entrapment of Protein Using Electrosprayed Poly(d,l‐lactide‐co‐glycolide) Microspheres with a Porous Structure for Sustained Release

The entrapment of a protein in porous poly(d,l ‐lactide‐co‐glycolide) (PLGA) microspheres is demonstrated through the closure of their outer surface pores for sustained delivery of the protein. The porous PLGA microspheres with less than 10 μm in size are prepared by electrospraying. Aqueous solutions containing fluorescein isothiocyanate‐dextran or bovine serum albumin (BSA) are penetrated into the inner pores as a result of vacuum treatment, and the outer surface pores of the porous PLGA microspheres are then closed using a solvent (dimethyl sulfoxide) to ensure entrapment of the macromolecules. Confocal microscopy images confirm the presence of a large amount of the macromolecules inside the porous structure. Circular dichroism spectroscopy and release analysis reveal that BSA is entrapped without denaturation and released in a sustained manner for a period of over 2 months, respectively.

     

In vitro evaluation of sustained release of risperidone‐loaded microspheres fabricated from different viscosity of PLGA polymers

The aim of this study was to fabricate different risperidone (RIS)–loaded poly(lactic‐co‐glycolic acid) microspheres (PLGA MS) with various viscosity of PLGA polymers and investigate the RIS release profiles. Risperidone‐loaded PLGA MS were fabricated via an oil/water–type emulsion solvent evaporation method, using ethyl acetate and benzil alcohol as the dispersed solvents. The PLGA (75:25) with the viscosity of 0.82, 0.93, and 1.10 dL/g was used in the fabrication. The morphology and the degradation of the RIS‐PLGA MS were investigated with a scanning electron microscope. The distribution states of RIS in the PLGA MS were studied with differential scanning calorimetry. The residual of ethyl acetate and benzil alcohol in the resulting MS was monitored with gas chromatography. The in vitro release profiles of RIS from PLGA MS were also studied. Scanning electron microscope photographs illustrated that the obtained PLGA MS were monodisperse spheres with an average diameter of 100 μm. Gas chromatography results suggested that no residual ethyl acetate was left in the final RIS‐PLGA MS, and the residue amount of benzil alcohol was about 1%. In vitro drug release profiles from the microsphere showed a substantially sigmoidal pattern of negligible initial burst up to 24 hours and minimal release (time lag) for 14 days. After the lag phase, slow release took a place up to 30 days, and then rapid release occurred sharply for about 2 weeks. The RIS release reached equilibrium around day 50. All release profiles showed the similar trend, and no significant difference was observed among these release profiles (P > .05). This phenomenon indicated that RIS‐loaded PLGA MS with the viscosity of 0.82 to 0.93 dL/g showed the optimal release behavior. However, no obvious differences were found among the MS obtained from the viscosity of 0.82 to 1.10 dL/g. These studies provided basis for the quality control in the industrial production of PLGA MS.     

Highly porous poly(lactide‐co‐glycolide) microparticles for sustained tiotropium release

In this study, porous poly(lactide‐co‐glycolide) (PLGA) microparticles with low mass density and large particle size were developed for chronic obstructive pulmonary disease treatment using anticholinergic drug (tiotropium). The porous PLGA microparticles were prepared by the water‐in‐oil‐in‐water (W₁/O/W₂) multi‐emulsion method using PLGA polymer and ammonium bicarbonate (as a porogen). Herein, soluble starch was incorporated in porous PLGA microparticles for long‐term tiotropium release. In vitro drug release studies determined that the rapid release of tiotropium from porous PLGA microparticles was reduced because of the high viscosity of the incorporated starch. Tiotropium release from porous PLGA microparticles continued up to 3 days. Furthermore, the inhaled microparticles showed longer drug residence in in vivo lung epithelium. Copyright © 2013 John Wiley & Sons, Ltd.     

Cross‐Platform Comparison of Therapeutic Delivery from Multilamellar Lipid‐Coated Polymer Nanoparticles

Significant efforts have been invested in finding a delivery system that can encapsulate and deliver therapeutics. Core–shell polymer‐lipid hybrid nanoparticles have been studied as a promising platform because of their mechanical stability, narrow size distribution, biocompatibility, and ability to co‐deliver diverse drugs. Here, novel core–shell nanoparticles based on a poly(lactic‐co‐glycolic acid) (PLGA) core and multilamellar lipid shell are designed, where the lipid bilayers are crosslinked between the two adjacent bilayers (PLGA‐ICMVs). The cross‐platform performance of the nanoparticles to other polymer‐lipid hybrid platforms is examined, including physicochemical characteristics, ability to encapsulate a variety of therapeutics, biocompatibility, and functionality as a vaccine delivery platform. Differential abilities of nanoparticle systems to encapsulate distinct pharmaceutics are observed, which suggest careful consideration of the platform chosen depending on the therapeutic agent and desired function. The novel PLGA‐ICMV platform herein demonstrates great potential in stably encapsulating water‐soluble agents and therefore is an attractive platform for therapeutic delivery.     

Development of New Microencapsulation Techniques Useful for the Preparation of PLGA Microspheres

Summary: Intensive efforts were made to develop an efficient, novel microencapsulation system useful to encapsulate a model drug, risperidone, to PLGA microspheres. Methyl dichloroacetate was used as a dispersed solvent for the first time, since it possessed excellent solvency power on PLGA and readily underwent ammonolysis. A dispersed phase composed of methyl dichloroacetate, risperidone, and PLGA was emulsified in an aqueous phase to form an O/W emulsion. Adding ammonia solution into the emulsion rapidly converted methyl dichloroacetate into water‐soluble dichloroacetamide and methanol. As a result, emulsion droplets were immediately transformed into hardened microspheres. The new microencapsulation system allowed us to make PLGA microspheres with a drug payload of >40 wt.‐% and attain almost complete encapsulation efficiencies. In summary, preparing an O/W emulsion and subjecting the emulsion to ammonolysis led to development of an efficient, novel microencapsulation system. It was anticipated that the new system could make it possible to load other bioactive materials into microspheres made of various types of hydrophobic polymers.

SEM micrographs of the external and internal morphology of PLGA/risperidone microspheres.     

Effects of lung surfactants on rifampicin release rate from monodisperse rifampicin-loaded PLGA microspheres

We have prepared inhalable and monodisperse poly(lactide-co-glycolide) (PLGA) microspheres targeting tubercle bacilli residing in alveolar macrophages. The effects of pulmonary surfactant on the rifampicin (RFP) release rate from RFP-loaded poly (lactide-co-glycolide) microspheres were studied. Also, those of their surface properties of RFP-loaded PLGA microspheres were studied. The RFP release from RFP/PLGA microspheres was accelerated by adsorption of pulmonary surfactant on the particle surface. The fastest RFP release rate was observed from pulmonary surfactant-adsorbed PLGA particles in pH 7.4 buffer solution compared with those in pH 4.0 buffer solution and saline solution. The slowest release rate was observed in the case when saline solution was used as dispersion phase of RFP/PLGA microspheres, although RFP release rate increased by the addition of pulmonary surfactant. From these results it is suggested that when RFP/PLGA microspheres are administrated by inhalation, the RFP release rates from the particles which are not taken up by alveolar macrophages and remain in the alveoli will be small. On the other hand, the RFP release rates and release amounts will be high after RFP/PLGA microspheres are taken up by alveolar macrophages existing in phagosomes, but they become relatively small after RFP/PLGA microspheres move into phagosome-lysosomes by the fusion of phagosomes with lysosomes. The absolute values of the electrophoretic mobility of PLGA microspheres increased by the adsorption of pulmonary surfactants on the surfaces of PLGA microspheres. By analyzing the experimental data using the soft-particle theory, it was indicated that the microspheres became 'softer' and the surface charge density of microspheres increases by the degradation. On the other hand, the surface of PLGA microspheres became harder and the electric charge density increased by the adsorption of pulmonary surfactant on the surfaces of PLGA microspheres. The changes in the surface charge density with degradation became larger by the adsorption of the lung surfactant on PLGA microsphere surfaces. It is considered that the changes in surface properties of PLGA microspheres affect their uptake efficiency by alveolar macrophage.     

Multi‐Colored Fibers by Self‐Assembly of DNA,Histone Proteins,and Cationic Conjugated Polymers

The development of biomolecular fiber materials with imaging ability has become more and more useful for biological applications. In this work, cationic conjugated polymers (CCPs) were used to construct inherent fluorescent microfibers with natural biological macromolecules (DNA and histone proteins) through the interfacial polyelectrolyte complexation (IPC) procedure. Isothermal titration microcalorimetry results show that the driving forces for fiber formation are electrostatic and hydrophobic interactions, as well as the release of counterions and bound water molecules. Color‐encoded IPC fibers were also obtained based on the co‐assembly of DNA, histone proteins, and blue‐, green‐, or red‐ (RGB‐) emissive CCPs by tuning the fluorescence resonance energy‐transfer among the CCPs at a single excitation wavelength. The fibers could encapsulate GFP‐coded Escherichia coli BL21, and the expression of GFP proteins was successfully regulated by the external environment of the fibers. These multi‐colored fibers show a great potential in biomedical applications, such as biosensor, delivery, and release of biological molecules and tissue engineering.     

Multi‐Colored Fibers by Self‐Assembly of DNA,Histone Proteins,and Cationic Conjugated Polymers

The development of biomolecular fiber materials with imaging ability has become more and more useful for biological applications. In this work, cationic conjugated polymers (CCPs) were used to construct inherent fluorescent microfibers with natural biological macromolecules (DNA and histone proteins) through the interfacial polyelectrolyte complexation (IPC) procedure. Isothermal titration microcalorimetry results show that the driving forces for fiber formation are electrostatic and hydrophobic interactions, as well as the release of counterions and bound water molecules. Color‐encoded IPC fibers were also obtained based on the co‐assembly of DNA, histone proteins, and blue‐, green‐, or red‐ (RGB‐) emissive CCPs by tuning the fluorescence resonance energy‐transfer among the CCPs at a single excitation wavelength. The fibers could encapsulate GFP‐coded Escherichia coli BL21, and the expression of GFP proteins was successfully regulated by the external environment of the fibers. These multi‐colored fibers show a great potential in biomedical applications, such as biosensor, delivery, and release of biological molecules and tissue engineering.     

Osmogen‐Mediated One‐Step Technique of Fabricating Hollow Microparticles for Encapsulation and Delivery of Bioactive Molecules

Microparticulate systems composed of biodegradable polymers, such as poly(d ,l ‐lactic‐co‐glycolic acid) (PLGA), are widely used for controlled release of bioactive molecules. However, the acidic microenvironment within these microparticles, as they degrade, has been reported to perturb the configuration of most encapsulated proteins. In addition, these polymer particles are also reported to suffer from unrealistically slow and incomplete release of proteins. To address these drawbacks, hollow PLGA microparticles are fabricated through a novel one‐step oil‐in‐water emulsion solvent evaporation technique, by capitalizing on the osmotic property of an osmogen. The effects of fabrication para­meters on particle size and morphology, i.e., volume space of hollow cavity and shell thickness, are also studied. These hollow microparticles are subsequently loaded with bovine insulin microcrystals. It is shown that insulin release profiles can be tuned by simply changing the amount of osmogen in the formulation. At the same time, these hollow microparticles are shown to be effective in maintaining the bioactivity of the encapsulated protein.

     

Preparation and characterization of PLGA particles for subcutaneous controlled drug release by membrane emulsification

Uniformly sized microparticles of poly(d,l-lactic-co-glycolic) (PLGA) acid, with controllable median diameters within the size range 40–140 μm, were successfully prepared by membrane emulsification of an oil phase injected into an aqueous phase, followed by solvent removal. Initially, simple particles were produced as an oil in water emulsion, where dichloromethane (DCM) and PLGA were the oil phase and water with stabiliser was the continuous phase. The oil was injected into the aqueous phase through an array type microporous membrane, which has very regular pores equally spaced apart, and two different pore sizes were used: 20 and 40 μm in diameter. Shear was provided at the membrane surface, causing the drops to detach, by a simple paddle stirrer rotating above the membrane. Further tests involved the production of a primary water in oil emulsion, using a mechanical homogeniser, which was then subsequently injected into a water phase through the microporous membrane to form a water in oil in water emulsion. These tests used a water-soluble model drug (blue dextran) and encapsulation efficiencies of up to 100% were obtained for concentrations of 15% PLGA dissolved in the DCM and injected through a 40 μm membrane.

Solidification of the PLGA particles was followed by removal of the DCM through the surrounding aqueous continuous phase. Different PLGA concentrations, particle size and osmotic pressures were considered in order to find their effect on encapsulation efficiency. Osmotic pressure was varied by changing the salt concentration in the external aqueous phase whilst maintaining a constant internal aqueous phase salt concentration. Osmotic pressure was found to be a significant factor on the resulting particle structure, for the tests conducted at lower PLGA concentrations (10% and 5% PLGA). The PLGA concentration and particle size distribution influence the time to complete the solidification stage and a slow solidification, formed by stirring gently overnight, provided the most monosized particles and highest encapsulation efficiency.     

An injectable and biomimetic multi‐phase nanocomposite for non‐invasive bone tissue engineering: fabrication and mechanistic evaluation

An injectable, non‐hardening nanocomposite bone graft has been developed using a combination of nanohydroxyapatite as bioactive and osseointegrative material; P‐15 peptide‐modified poly(lactic‐co‐glycolic acid) (PLGA) microspheres as biomimetic and osteoinductive agent; and PLGA–poly(ethylene glycol) (PEG)–PLGA as a carrier gel. Increase in lactic acid/glycolic acid ratio of PLGA–PEG–PLGA resulted in stronger gels with a wider gelation window. Addition of 2.5‐fold nanohydroxyapatite resulted in significant changes in injectability (3.5‐fold force of injection), swelling characteristics (2.5 times swelling index), rheological (shear viscosity from 2.1 × 10¹ Pa s for NC3_700 to 1.5 × 10⁶ Pa s for NC3_73.52 and from 3.9 × 10² Pa s for NC8_700 to 3.76 × 10⁶ Pa s for NC8_732; an increase in elasticity at the level of 1–1000 kPa), and thermal properties of the nanocomposites. A mechanistic study showed that nanohydroxyapatite exhibits a high degree of association with the gel and interferes with its gelation owing to changes in hydrogen bonding interactions between C=?O of polymer chains and P–OH groups of nanohydroxyapatite with water molecules of the gel. A schematic was developed demonstrating changes in bonding interactions among constituent phases with respect to nanohydroxyapatite content emphasizing the importance of material interactions while fabricating multi‐phase nanocomposites for various biomedical applications. Copyright © 2017 John Wiley & Sons, Ltd.     

Surfactant‐Free Emulsion‐Based Preparation of Redox‐Responsive Nanogels

A surfactant‐free emulsion‐based approach is developed for preparation of nanogels. A water‐in‐oil emulsion is generated feasibly from a mixture of water and a solution of disulfide‐containing hyperbranched PEGylated poly(amido amine)s, poly(BAC2‐AMPD1)‐PEG, in chloroform. The water droplets in the emulsion are stabilized and filled with poly(BAC2‐AMPD1)‐PEG, and the crosslinked poly(amido amine)s nanogels are formed via the intermolecular disulfide exchange reaction. FITC‐dextran is loaded within the nanogels by dissolving the compound in water before emulsification. Transmission electron microscopy and dynamic light scattering are applied to characterize the emulsion and the nanogels. The effects of the homogenization rate and the ratio of water/polymer are investigated. Redox‐induced degradation and FITC‐dextran release profile of the nanogels are monitored, and the results show efficient loading and redox‐responsive release of FITC‐dextran. This is a promising approach for the preparation of nanogels for drug delivery, especially for neutral charged carbohydrate‐based drugs.

     

N‐Boc‐Histidine‐Capped PLGA‐PEG‐PLGA as a Smart Polymer for Drug Delivery Sensitive to Tumor Extracellular pH

A pH‐sensitive polymer was synthesized by introducing the N‐Boc‐histidine to the ends of a PLGA‐PEG‐PLGA block copolymer. The synthesized polymer was confirmed to be biodegradable and biocompatible, well dissolved in water and forming micelles above the CMC. DOX was employed as a model anticancer drug. In vitro drug release from micelles of N‐Boc‐histidine‐capped PLGA‐PEG‐PLGA exhibited significant difference between pH = 6.2 and pH = 7.4, whereas DOX release from micelles composed of un‐capped virgin polymers was not significantly sensitive to medium pH. Uptake of DOX from micelles of the new polymer into MDA‐MB‐435 solid tumor cells was also observed, and pH sensitivity was confirmed. Hence, the N‐Boc‐histidine capped PLGA‐PEG‐PLGA might be a promising material for tumor targeting.

     

Effect of polyethylene glycol on preparation of rifampicin-loaded PLGA microspheres with membrane emulsification technique

Monodisperse poly(lactide-co-glycolide) (PLGA) microspheres containing rifampicin (RFP), anti-tubercle drug, as hydrophobic model drug were prepared by solvent evaporation method with a membrane emulsification technique using Shirasu Porous Glass (SPG) membranes. Five kinds of rifampicin-loaded PLGA (RFP/PLGA) microspheres with different sizes were prepared by changing pore size of the membranes. Effect of polyethylene glycol (PEG) added to polyvinyl alcohol (PVA) solution (continuous phase) upon the monodispersity of microspheres was studied. PEG was used as a stabilizer for microspheres dispersing in PVA solution. The most suitable molecular weight of PEG as a stabilizer was 20,000. RFP/PLGA microspheres prepared with PEG20000 were apparently more uniform than those prepared without PEG. The yield of RFP/PLGA microspheres was 100%. The initial burst observed in the release of RFP from RFP/PLGA microspheres was suppressed by the addition of PEG.     

Thermoresponsive hydrophobic copolymer grafted on agar microspheres for all‐aqueous bioseparations

Agar microspheres were prepared by water–oil emulsification and cross‐linked under alkaline condition. The thermoresponsive hydrophobic copolymer, poly(N‐isopropylacrylamide‐co‐lauryl methacrylate‐co‐acrylamide), was grafted on the agar microspheres via atom transfer radical polymerization. The agar microspheres grafted with copolymers were characterized by light microphotography, elemental analysis, Fourier transform infrared spectroscopy, scanning electron microscopy, and X‐ray photoelectron spectroscopy. The chain lengths and hydrophobic monomer ratio of the grafting linear polymer had significant effects on the hydrophobicity and adsorption capacity of agar microspheres at different temperatures. The thermoresponsive microspheres were used for separation of proteins and showed binding and release behavior by change of temperatures without change in mobile phase composition. Thus, we suggest thermoresponsive agar microspheres as an alternative separation media for all‐aqueous bioseparations.     

One‐step synthesis of poly(lactic‐co‐glycolic acid)‐g‐poly‐1‐vinylpyrrolidin‐2‐one copolymers

The radical polymerization of 1‐vinylpyrrolidin‐2‐one (NVP) in poly(lactic‐co‐glycolic acid) (PLGA) 50:50 at 100 °C leads to amphiphilic PLGA‐g‐PVP copolymers. Their composition is determined by FT‐IR spectroscopy. Thermogravimetric analyses agree with FT‐IR determinations. Saponification of the PLGA‐g‐PVP polyester portion allows isolating the PVP side chains and measuring their molecular weight, from which the average chain transfer constant (C_T) of the PLGA units is estimated. The MALDI‐TOF spectra of PVP reveal the presence at one chain end of residues of either glycolic acid‐ or lactic acid‐ or lactic/glycolic acid dimers, trimers and one tetramer, the other terminal being hydrogen. This unequivocally demonstrates that grafting occurred. Accordingly, the orthogonal solvent pair ethyl acetate—methanol, while separating the components of PLGA/PVP intimate mixtures, fails to separate pure PVP or PLGA from the reaction products. All PLGA‐g‐PVP and PLGA/PLGA‐g‐PVP blends, but not PLGA/PVP blends, give long‐time stable dispersions in water. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1919–1928     

Fast reversed‐phase liquid chromatographic separation of proteins by flow‐through poly(styrene‐co‐divinylbenzene) microspheres

With the explosive growth of the bioscience and biopharmaceuticals, the demand for high efficient analysis and separation of proteins is urgent. High‐performance liquid chromatography is an appropriate technology for this purpose, and the stationary phase is the kernel to the separation efficiency. In this study, flow‐through poly(styrene‐co‐divinylbenzene) microspheres characteristic of the binary pores, i.e. flow‐through pores and mesopores, were synthesized; this special porous structure would benefit the convective mass transfer while guarantee the high specific surface area. Owing to the hydrophobic nature, poly(styrene‐co‐divinylbenzene) microspheres were suitable as the reversed‐phase stationary phase for separation of proteins. For the high permeability of the poly(styrene‐co‐divinylbenzene) microspheres packed column, fast separation of the studied six proteins in ～2 min was achieved. The recoveries of studied proteins were acceptable in the range of 79.0–99.4%. The proposed column had good pH stability of 1–13 and repeatability. Moreover, the column was applied for egg white fast separation, further demonstrating its applicability for complex bio‐sample separation. The flow‐through poly(styrene‐co‐divinylbenzene) microspheres were promising for fast separation of large molecules.