Advances in Colloid and Interface Science 209 (2014) 1–7
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Protein microcapsules: Preparation and applications Maheshkumar Jaganathan a, D. Madhumitha b, A. Dhathathreyan b a b
Chemical Lab., CSIR-CLRI, Adyar, Chennai 600020, India Biophysics Lab., CSIR-CLRI, Adyar, Chennai 600020, India
a r t i c l e
i n f o
Available online 19 December 2013 Keywords: Protein Capsules Multiwalled Biomedicine Multicore Covalent Non covalent
a b s t r a c t Liposomes and polymerosomes generally represent the two most widely used carriers for encapsulating compounds, in particular drugs for delivery. While these are well established carriers, recent applications in biomedicine and food industry have necessitated the use of proteins as robust carriers that are stable under extreme acidic and basic conditions, have practically no toxicity and are able to withstand high shear force. This review highlights the different methods for using proteins as encapsulating materials and lists some biomedical applications of the microcapsules. The advantages and limitations in the capsules from the different preparation routes are enumerated. © 2013 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Methods of preparation . . . . . . . . . . . . . . . . . . . 2.1. Capsules using covalent interactions . . . . . . . . . . 2.1.1. LbL and other modified techniques of LbL . . . . 2.1.2. LbL method combined with post functionalization 2.2. Non covalent methods of microcapsule preparation . . . 2.3. Protein microcapsules using ‘freeze–thaw’ technique . . . 3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Scientists have long been intrigued by the possibilities of controlling the release of an active ingredient by encapsulating it. The necessity to find biomacromolecular drugs has given rise to the development of new methods of encapsulating the drugs using hollow structures (capsules) of biomolecules. Liposomes traditionally from phospholipids represent the standard model for biological capsule [1] and are used widely in cancer therapy. However they need to be chemically modified by PEGylation to stabilize them against immune response [2]. Polymerosomes are another class of aggregated structures and are designed either from pure polymers or from polymer/peptide units grafted with methoxy polyethylene glycol [3]. All these systems have the common feature of amphiphilicity of the compounds involved. These encapsulating systems find wide applications in pharmaceutical industry as controlled drug release devices, for therapy and for 0001-8686/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cis.2013.12.004
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immobilizing other macromolecules [4]. In recent times, new microcapsules based on biopolymers like proteins are being introduced as viable alternative to the polymerosomes or liposomes. A protein has to be cross-linked or stabilized using various methods in order to achieve sustained or controlled release properties. Easily available and often biocompatible, biopolymers play an increasing role among the materials available to constitute the frame of microparticles. Furthermore, the functional groups of proteins are available for chemical modifications needed for encapsulation by chemical methods. However the different methods that are currently available have their own limitations with some of them suffering from toxicity issues. Recently, genome nucleic acids encapsulated in viral capsid proteins have been used for viral cell transformation [5]. This review highlights the different techniques used to form protein capsules that can act as vehicle for encapsulating drugs or even as templates to prepare new materials. In the preparation of protein microcapsules, a wide variety of protein aggregates are encountered ranging in
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size and characteristics (e.g., soluble or insoluble, covalent or noncovalent, reversible or irreversible) and span a broad size range, from small oligomers (nanometers) to insoluble micron-sized aggregates that can contain millions of monomer units. These aggregates result from various kinds of stress such as agitation and exposure to extremes of pH, temperature, ionic strength, or various interfaces (e.g., air–liquid interface). Therefore, the preparation of the microcapsules of protein needs to be carefully characterized and controlled during development, manufacture, and subsequent storage of a drug substance and formulated product. Microcapsules in general refer to spherical microparticles and “microcapsules” happen to be a sub category of the microparticles which have a core surrounded by a material which is distinctly different from that of the core. The core may be solid, liquid, or even gas [6]. Till now several approaches have been used to design microcapsules and a number of applications have been reported [7–19]. 2. Methods of preparation In general, the term “microcapsule” is defined, as a spherical particle with the size varying between 50 nm to 2 mm containing a core substance. However, though the word capsule implies a core and shell structure, the term microcapsules can also include membrane enclosed particles or droplets but also dispersion in solid matrix lacking a distinctive external wall phase as well as intermediate types and depending on the method of preparation, the various types of capsules can be obtained (Fig. 1.). The general strategies for formation of protein capsules usually are from two different routes: covalent and noncovalent interactions. Covalent interactions generally increase the mechanical strength of the capsules as has been demonstrated in the layer by layer method of preparation which uses glutaraldehyde [20–22]. However, the use of covalent interactions to form the capsules has its own limitations in that it results in permanent bonds which seem to trigger larger assemblies of the capsules and in some instances can trigger cytotoxicity [23–25]. A viable alternative is to use noncovalent interactions (e.g., with amphiphilic molecules, electrostatic interactions, hydrogen bonding and hydrophobic interactions). These include formation of hydrogels [26–30], particles formed between proteins and ligands, polymers and colloidal systems.
Using the hydrophobic/hydrophilic interface, Suslick et al. used ultrasonication on proteinaceous materials in which a water insoluble liquid was enclosed and demonstrated that high concentrations with narrow size distributions of the microcapsules can be formed. The scheme for the preparation of such assemblies is presented in Fig. 2. They also showed that this involves both emulsification and a chemical cross-linking of protein molecules through disulfide bond formation by sonochemically generated superoxide [31]. Interfacial tension between water and an organic phase was used by Liu et al. to prepare encapsulated assemblies of proteins on organic droplets [32]. These oil/water interfaces while stabilizing the protein at the interface, however have an inherent disadvantage in that the oil droplets are not suitable to dissolve water-soluble guest biopolymers. Morikawa et al. have used ionic liquid–water interface to encapsulate proteins in biopolymers [33]. Here surface-modified protein microcapsules spontaneously form at ambient temperature. The size and rate of formation depend on the type of protein and also on the charge density on the surface of the protein which can be regulated by suitable choice of the anions or cations of the ionic liquid. Following section pertains to use of LbL technique and modification of this technique in the preparation of protein capsules.
2.1. Capsules using covalent interactions 2.1.1. LbL and other modified techniques of LbL Design of microcapsules based on electrostatic interactions has been carried out by layer by layer adsorption (LbL) of polyelectrolytes in many examples including polypeptides built on colloidal templates. This method developed by Decher et al. uses sequential deposition of oppositely charged layers on a template that can then form polyelectrolyte shells [34,35]. Here the shell thickness can be controlled and with suitable triggers can be used to release materials [36,37]. Capsules prepared using the LbL technique have been made using a variety of cargo molecules that include polymers, low molecular weight compounds, and enzymes [38–43]. Zelikin et al. have used a facile method to encapsulate both single as well as double stranded DNA in nanoengineered, degradable polymer
Fig. 1. Scheme showing different methods of preparation for protein microcapsules.
M. Jaganathan et al. / Advances in Colloid and Interface Science 209 (2014) 1–7
Fig. 2. Schematic illustration of fabrication of protein microcapsule via oil/water emulsion using sonication.
microcapsules and have used amine functionalized silica particles as template in which the DNA was adsorbed (Fig. 3.). Then a sequential deposition of thiolated PMA (PMA SH) and poly(vinylpyrrolidone) have been used to form multilayers; followed by cross-linking of the thiol groups of the PMASH in the multilayers into disulfide linkages. Finally the sacrificial SiO + 2 particles have been removed. The DNA has been shown to retain its functionality and structural integrity [44]. In all the capsules formed from the LbL method, the cytotoxicity of synthetic polymer shells often arising from the use of cationic polyelectrolytes seems to be a major disadvantage. De Koker et al. overcame the limitations of toxicity in LbL technique, by using polyelectrolyte capsules consisting of dextran sulfate and polyL-arginine in which antigen proteins were loaded and they showed that such capsules degrade in dendritic cells through proteases and subsequently release the antigen [45]. Temmermann and co-workers [46] evaluated the encapsulation efficiency of such microcapsules and showed that the efficiency is strongly dependent on both the charge and the molecular weight of the protein and on the number of polyelectrolyte bilayers the microcapsules consist of. In the context of use of the microcapsules as carrier for biomedical applications, it is necessary to prepare microcapsules having low toxicity, with robust shells that are stable under extreme acidic and basic conditions, and can withstand high shear force. Ye et al. have used chemically modified silk fibroin to form microcapsules using LbL technique. Here they applied cationic silk-poly- L-lysine (SF-PL) or anionic silk-poly-L-glutamic acid (SF-PG) that allowed the formation of stable
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ultrathin shell microcapsules with a dramatic increase in swelling, thickness, and microroughness at extremely acidic (pH b 2.5) and basic (pH N 11.0) conditions without much disintegration. The additional advantage with these microcapsules is that they undergo remarkable softening with a reduction in Young's modulus by more than 1 order of magnitude due to the swelling, stretching, and increase in material porosity which finds applications in bioengineering [47]. Zhou et al. [48] showed that the poly-L-lysine can be conjugated as a pro-drug of cisplatin (II). Here a Pt (IV) complex with a carboxyl group was synthesized and conjugated to the side chains of poly-L-lysine (PLL), resulting in a polypeptide-drug conjugate, PLL-Pt (IV). PLL-Pt (IV) was then assembled with poly-L-glutamic acid (PGA) through LbL technique on a colloidal support into biodegradable, controllably drugloaded, and environmentally responsive polypeptide capsules. Platinum release from the capsules was found to be greatly enhanced under low pH and reductive conditions. Lee et al. demonstrated the use of LbL designed hyaluronic acid (HA)—poly-L-lysine (PLL) adsorbed systems in which the core removal was carried out by the reducing agent dithiothreitol at neutral pH [49]. The HA/PLL multilayers on the shell were chemically cross-linked via carbodiimide chemistry and showed enhanced physical stability against freeze–thaw cycles and acidic pH conditions. These microcapsules were then used to encapsulate bovine serum albumin (BSA) which could be released using pH gradients or by adding an HA digesting enzyme (hyaluronidase) in the incubation medium.
2.1.2. LbL method combined with post functionalization Improving on the LbL technique Ochs et al. have used an elegant combination of LbL with click chemistry wherein initially poly-L-lysine (PLL) and poly-L-glutamic acid (PGA) have been modified with alkyne (ALK) and azide (AZ) moieties [50]. The capsules obtained for (PLL-AZ/PLL-Alk) multilayer films assembled at pH 5/9 as well as for (PGA-AZ/PGA-ALK) assembled at pH 4 were stable in the range of pH 2 to 11 and exhibited pH-responsive swelling/shrinking. The advantage with this method is that typically individual layers or the capsule surfaces made from biodegradable materials, can be post-functionalized by the use of either excess click groups or other functionality in the film and offer a promising approach towards the design of novel biodegradable vehicles for targeted drug delivery.
Fig. 3. Scheme for encapsulation of DNA in nano-engineered degradable microcapsules.
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Becker and co-workers showed that capsules prepared using LbL technique can be used to deliver small interfering RNAs (siRNA) in prostate cancer cells [51]. They used thiol modified polymethacrylic acid poly-L-lysine (PMASH-PLL) microcapsules. Here a nanoporous particle template was used into which PLL was infiltrated prior to PMASH multilayer assembly. The siRNA cargo was then post loaded into these preformed microcapsules by diffusion through the multilayer film and complexation by the PLL. Correia and co-workers in a recent paper reported on the use of multilayered hierarchical capsules for facilitating cell adhesion sites. Using a simple but elegant method of using liquefied capsules with assembly of poly-L-lysine, alginate, and chitosan they encapsulated surface functionalized poly-L-lactic acid (PLLA) and showed that these can provide support for cellular functions of anchorage dependent cells [52]. The optimal use of bioactivity of protein microcapsules has been demonstrated by Wang et al. and they used the Michael addition and Schiff base reactions in between two layers to form protein microcapsules having a robust but flexible shell [53]. They initiated the process by using a protein-doped CaCO3 template synthesized via co precipitation, which was then coated with a catecholcontaining alginate (AlgDA) layer. The templates were then exposed to ethylenediamine tetraacetic acid disodium (EDTA) solution to dissolve CaCO3. During CaCO3 dissolution, the generated CO2 gas pushes protein molecules moving to the AlgDA layer, and thereby reactions proceed, forming the shell of protein microcapsules. A scheme of the formation of these capsules is presented in Fig. 4. 2.2. Non covalent methods of microcapsule preparation For the first time, Humblet Hua et al. have used protein fibrils to enhance the mechanical strength of the microcapsules. By using different proteins like lysozyme and ovalbumin, they have shown that the variation in the flexibility of the protein fibrils can change the mechanical properties of the capsules. In their experiments they used various ratios of ovalbumin or lysozyme having positive charges with high methoxyl pectin which is anionic and showed that this method does not require any extra salts to be added [54]. Duongthingoc and colleagues using spray dried whey protein close to isoelectric point have shown the formation of an early crust in the microcapsules. Here the protein in the molten globular state creates a cohesive network that has been used to encapsulate yeast cells [55]. Puga and co-workers have demonstrated that pectin-coated chitosan microgels crosslinked on superhydrophobic surfaces can be used to encapsulate 5-fluorouracil which could then be used to deliver the drug onto cancer cells [56]. In the food industry, specifically in the manufacture of nutraceuticals or functional food, the use of spray drying for the production of anhydrobiotics has gained much importance, mainly due to cost efficiencies, enhanced product shelf life. Behboudi-Jobbehdar et al. used a composite of whey protein, maltodextrin and D-glucose carrier to encapsulate the thermo sensitive probiotic lactobacilli strains Lactobacillus acidophilus applying the spray drying technique [57]. The microcapsules obtained were spherical in shape with few surface cavities which seemed to stabilize the lactobacilli.
Mao et al. in their work have demonstrated elegantly the use of mixed protein systems as emulsions to deliver carotenoids [58]. In this work, oil in water emulsions of β-lactoglobulin, lactoferrin and their mixed systems have been analyzed for their suitability to stabilize and deliver β-carotene. The method seems to suggest a simple delivery vehicle for bioactive components. Matalanis and McClements have demonstrated the use of hydrogel microspheres fabricated from a phase separated mixture of pectin, caseinate, and emulsified oil to form an oil-in-water-in-water (O/W1/W2) emulsion which has been then acidified and finally cross-linked with transglutaminase. Such microspheres trapped within bioactive molecules have been used efficiently to deliver lipophilic agents. Using various proportions of the continuous biopolymer phase, the authors could optimize the yield as well as the performance of the microspheres [59]. Chen and co-workers used whey protein isolate and sodium caseinate to encapsulate lipophilic bioactive components like fish oil, phytosterols and limonene and preserve the flavor and odor. The thickness of the protein walls of the capsules could be regulated by the ratio of the whey protein to the caseinate [60]. In the delivery of DNA for therapeutics as well as in studies on hybridization often carriers are used that can deliver the nucleotides specifically. Hardin et al. used gelatin microcapsules which were initially fabricated using microfluidics [61]. Microcapsules were loaded with the 15 base long secondary DNA targets, then capped with a polyelectrolyte bilayer and finally with a monolayer of polystyrene microcapsules. When the capsules were warmed to 37 °C, secondary DNA targets were released from the gelatin template and then displaced the shorter, original hybridization partners on the polystyrene microcapsules. Mazzitelli et al. in a two-step gelation process have used pectin and gelatin [62]. Here, the first thermal step helps in gelation of gelatin followed by an ionic one where gel structure of pectin occurs. The viscoelastic properties of these gel combinations have been optimized to make an efficient transdermal patch. These patches have been used as transdermal system to deliver testosterone and the in vitro drug release profiles have been analyzed. Shimokawa et al. have used gelatin– water–lower alcohol systems in different proportions to form microcapsules by coacervation method and have analyzed the drug release profile from the different ternary systems [63]. Normally in the development of drugs using peptides and proteins the difficulty lies in their slow clearance in the liver and other body tissues by proteolytic enzymes. Hence there is a difficulty to administer these drugs through regular injections. Zheng et al. have designed and applied a biodegradable and redoxresponsive submicron capsules using the LbL technique with poly-Laspartic acid and chitosan for transmucosal delivery [64]. Erokhina and co-workers have recently reported on a novel method of using collagen type I microcapsules and the release mechanism from these capsules has been carried out using a biological stimulus from an over-expressed collagenolytic matrix metalloproteinase I (MMPI) [65]. This method requires no external labels to study the release kinetics. Here the release of any drug through specific pore opening on the microcapsule surface can be regulated. Thus, this allows control of the release time and a variety of drugs of different sizes can be released.
Fig. 4. Scheme for protein capsules through template mediated interfacial reaction.
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Most of the methods described so far are based on some form of chemical microencapsulation methods. Most application scientists however associate the chemical methods with strong operating conditions and feel it suffers from non-green technologies. 2.3. Protein microcapsules using ‘freeze–thaw’ technique Among the noncovalent methods for preparing the protein microcapsules already mentioned above, most methods use amphiphilicity of the molecules for effecting hydrophobic interactions or even to create an interface. However Daamen et al. in 2007 reported for the first time the use of a simple method to prepare biocapsules at ambient or low temperatures from a wide class of biomolecules. This method is not dependent on the amphiphilicity of the biomolecules and uses a combination of freezing, annealing, and lyophilization procedure [66]. Microcapsules of extra cellular matrix protein elastin which is fibrous, in the size range 200 nm to 10 μ, have been designed using a soluble form of the protein in the above protocol. Microcapsules of a variety of macromolecules, including serum albumin, a globular protein, type I collagen (280 kDa) a fibrous protein and heparin as an example of a highly negatively charged polysaccharide have been prepared by this method. Using this technique, they demonstrated that this method can encapsulate both hydrophilic and lipophilic compounds, and can be prepared in large quantities. They described the mechanism of capsule formation by suggesting a three step process of microphase separation by fast-freezing, structural rearrangement by annealing to room temperature followed by the creation of a lumen by lyophilization. In their work, they had suggested the use of droplets of the protein which can be fast-frozen in liquid nitrogen and capsules prepared in high quantities. Using this droplet technique, Daisy Rani et al. prepared microcapsules of fibrinogen, a fibrous protein which was used as template to form nano clusters of nickel oxide [67]. Here the capsules of complexes of nickel–fibrinogen formed stable coatings on solid substrates which were then subjected to heating to form nickel hydroxide and subsequently nickel oxide. Madhumitha and Dhathathreyan have prepared microcapsules of bovine serum albumin (BSA) in the size range 0.2 to 0.3 μ. High resolution transmission electron micrograph of the capsules prepared at pH = 7.5 is shown in Fig. 5 [68]. The samples for TEM shown in the study do not have any external staining agent. In this technique capsules of the protein at pH 7.5 were prepared by dissolving the protein in phosphate buffer and introduced as droplets in a container with liquid nitrogen which was then slowly brought to − 20 °C for 2 h (annealing step) after which it was slowly brought to 5 °C and then crosslinked with 0.1% glutaraldehyde. Solutions of
Fig. 5. High resolution TEM of bovine serum albumin capsules at pH = 7.5.
Fig. 6. Scheme for preparation of protein microcapsules using the ‘freeze–thaw’ technique.
the protein at different pH (pH ranging from 3.5 to 8.5) were used in the above method and stable capsules of the protein at all pH could be obtained. A scheme for this method is shown in Fig. 6. Maheshkumar and Dhathathreyan used the ‘droplet technique’ and designed hemoglobin capsules in the size range of 0.1 to 0.3 μ which were then stabilized as films at air/buffer and solid/air interface [69]. After the freeze–thaw cycle they were then crosslinked with 0.1% glutaraldehyde. Particle size analysis of the capsules carried out using dynamic light scattering showed a major population (69%) with an average size of about 0.1–0.2 μ and populations of 1 to 2 μ (18%) and the rest in the range 3–4 μ. Fig. 7(a) and (b) shows the transmission electron micrographs of the capsules at pH 5.5 and 7.5 respectively. The samples formed were spread as films at the interface by spreading them on the buffer of pH 6.8 (pI of the protein). This method of stabilizing different protein films by spreading them on buffers at the isoelectric point has been used by Dhathathreyan and co-workers [70–72]. The average surface area versus surface pressure isotherms for these spread films showed stable films at the air/buffer interface. The dilational rheology of the capsules of the protein spread in a Langmuir was studied using oscillatory compressions of the film and by analyzing the surface pressure and the phase shift between the surface tension signal and the surface area. The protein capsules are composed of both hydrophobic and hydrophilic segments in appropriate proportion, and at the air/water interface they exhibit properties of a Janus-type particle with hydrophilic groups oriented towards the bulk water and the hydrophobic moieties organizing towards the air interface. The local interactions due to the asymmetry experienced by the water molecules at the protein/water interface and the changes in hydrophobic interactions seem to drive the assembly process. Due to strong stabilizing effect of the lateral electrostatic repulsions, such monolayer when compressed forms close-packed hexagonal structure, and can be easily transferred onto a solid substrate with the Langmuir–Blodgett technique. The results from their work suggest that stable capsules of pure proteins can be designed as films that can find biomedical applications and can be used to encapsulate small peptides and some lipophilic compounds. The micrographs in Fig. 7 suggest that this technique results in multicore microcapsules which can then be used either to diffuse or dissolve the contents and then release them. The model of formation of the protein capsules is complex and involves many processes such as capsule–interface interactions, protein orientations on the surface, lateral protein–protein interactions, bound water to free water ratios on the surface. Further secondary structural analysis and mass spectroscopy showed that the capsules had hemoglobin in the native state in tetrameric form suggesting that half life can be increased and corresponding toxicity also can be controlled. Duan et al. reported a simple and controllable method of using a CaCO3 particle as template to design Hb spheres with a high loading content in combination with the LbL technique [73]. The surface of the
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Fig. 7. Transmission electron micrographs of hemoglobin at (a) pH = 5.5; (b) pH = 7.5.
Hb spheres was then chemically modified by biocompatible polyethylene glycol to protect and stabilize the system. Using the freeze–thaw technique, Maheshkumar and Dhathathreyan have used the microcapsules of Cytochrome c (cyt c) prepared to model fibril formation. Fig 8(a) shows the TEM micrograph of the protein capsules and Fig. 8(b) shows the particle size analysis of the capsules. The capsules of cytochrome c obtained were subjected to repeated compression and expansion at air/buffer interface and the microcapsules are elastic up to some pressure beyond which they start to fuse. Till the point of fusion the conformation of the protein in the capsules remains fairly unchanged. However on fusion, the protein seems to undergo dramatic change in the secondary structure and transits to a more beta sheet like structure from the original helical structure.
3. Conclusion In summary, several techniques exist for preparation of robust assembly of protein microcapsule using different driving forces like amphiphilicity, electrostatic interactions, hydrogen bonding and hydrophobic interactions at fluid–fluid interfaces using both covalent and non covalent interactions between the biopolymer and either the active matter or any other functionality. The non covalent method seems to result in microcapsules with multicore structures for the proteins. The surface of the protein microcapsules can be easily modified by functional materials such as quantum dots and these can be conjugated with other molecules. By using the same strategy, numerous functional materials can be displayed on the protein microcapsules for a variety of biotechnological and biomedical applications. Significant strides have been made in the design and application of proteinaceous materials in microcapsules as carriers for food, nutraceuticals, dyes and for drug encapsulation. Improving the stability, sizes and overall properties of the capsules for immobilizing bioactive molecules is of great interest and more research needs to be carried out in this direction. Acknowledgments One of the authors MJ would like to thank DST-INSPIRE for fellowship. The authors would like to thank the Council of Scientific and Industrial Research (12th plan supra-institutional project-STRAIT-A/2013/BPY/ CSC0201/1035) for funding. References [1] [2] [3] [4]
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Contents lists available at ScienceDirect
Advances in Colloid and Interface Science journal homepage: www.elsevier.com/locate/cis
Protein microcapsules: Preparation and applications Maheshkumar Jaganathan a, D. Madhumitha b, A. Dhathathreyan b a b
Chemical Lab., CSIR-CLRI, Adyar, Chennai 600020, India Biophysics Lab., CSIR-CLRI, Adyar, Chennai 600020, India
a r t i c l e
i n f o
Available online 19 December 2013 Keywords: Protein Capsules Multiwalled Biomedicine Multicore Covalent Non covalent
a b s t r a c t Liposomes and polymerosomes generally represent the two most widely used carriers for encapsulating compounds, in particular drugs for delivery. While these are well established carriers, recent applications in biomedicine and food industry have necessitated the use of proteins as robust carriers that are stable under extreme acidic and basic conditions, have practically no toxicity and are able to withstand high shear force. This review highlights the different methods for using proteins as encapsulating materials and lists some biomedical applications of the microcapsules. The advantages and limitations in the capsules from the different preparation routes are enumerated. © 2013 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Methods of preparation . . . . . . . . . . . . . . . . . . . 2.1. Capsules using covalent interactions . . . . . . . . . . 2.1.1. LbL and other modified techniques of LbL . . . . 2.1.2. LbL method combined with post functionalization 2.2. Non covalent methods of microcapsule preparation . . . 2.3. Protein microcapsules using ‘freeze–thaw’ technique . . . 3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Scientists have long been intrigued by the possibilities of controlling the release of an active ingredient by encapsulating it. The necessity to find biomacromolecular drugs has given rise to the development of new methods of encapsulating the drugs using hollow structures (capsules) of biomolecules. Liposomes traditionally from phospholipids represent the standard model for biological capsule [1] and are used widely in cancer therapy. However they need to be chemically modified by PEGylation to stabilize them against immune response [2]. Polymerosomes are another class of aggregated structures and are designed either from pure polymers or from polymer/peptide units grafted with methoxy polyethylene glycol [3]. All these systems have the common feature of amphiphilicity of the compounds involved. These encapsulating systems find wide applications in pharmaceutical industry as controlled drug release devices, for therapy and for 0001-8686/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cis.2013.12.004
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immobilizing other macromolecules [4]. In recent times, new microcapsules based on biopolymers like proteins are being introduced as viable alternative to the polymerosomes or liposomes. A protein has to be cross-linked or stabilized using various methods in order to achieve sustained or controlled release properties. Easily available and often biocompatible, biopolymers play an increasing role among the materials available to constitute the frame of microparticles. Furthermore, the functional groups of proteins are available for chemical modifications needed for encapsulation by chemical methods. However the different methods that are currently available have their own limitations with some of them suffering from toxicity issues. Recently, genome nucleic acids encapsulated in viral capsid proteins have been used for viral cell transformation [5]. This review highlights the different techniques used to form protein capsules that can act as vehicle for encapsulating drugs or even as templates to prepare new materials. In the preparation of protein microcapsules, a wide variety of protein aggregates are encountered ranging in
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size and characteristics (e.g., soluble or insoluble, covalent or noncovalent, reversible or irreversible) and span a broad size range, from small oligomers (nanometers) to insoluble micron-sized aggregates that can contain millions of monomer units. These aggregates result from various kinds of stress such as agitation and exposure to extremes of pH, temperature, ionic strength, or various interfaces (e.g., air–liquid interface). Therefore, the preparation of the microcapsules of protein needs to be carefully characterized and controlled during development, manufacture, and subsequent storage of a drug substance and formulated product. Microcapsules in general refer to spherical microparticles and “microcapsules” happen to be a sub category of the microparticles which have a core surrounded by a material which is distinctly different from that of the core. The core may be solid, liquid, or even gas [6]. Till now several approaches have been used to design microcapsules and a number of applications have been reported [7–19]. 2. Methods of preparation In general, the term “microcapsule” is defined, as a spherical particle with the size varying between 50 nm to 2 mm containing a core substance. However, though the word capsule implies a core and shell structure, the term microcapsules can also include membrane enclosed particles or droplets but also dispersion in solid matrix lacking a distinctive external wall phase as well as intermediate types and depending on the method of preparation, the various types of capsules can be obtained (Fig. 1.). The general strategies for formation of protein capsules usually are from two different routes: covalent and noncovalent interactions. Covalent interactions generally increase the mechanical strength of the capsules as has been demonstrated in the layer by layer method of preparation which uses glutaraldehyde [20–22]. However, the use of covalent interactions to form the capsules has its own limitations in that it results in permanent bonds which seem to trigger larger assemblies of the capsules and in some instances can trigger cytotoxicity [23–25]. A viable alternative is to use noncovalent interactions (e.g., with amphiphilic molecules, electrostatic interactions, hydrogen bonding and hydrophobic interactions). These include formation of hydrogels [26–30], particles formed between proteins and ligands, polymers and colloidal systems.
Using the hydrophobic/hydrophilic interface, Suslick et al. used ultrasonication on proteinaceous materials in which a water insoluble liquid was enclosed and demonstrated that high concentrations with narrow size distributions of the microcapsules can be formed. The scheme for the preparation of such assemblies is presented in Fig. 2. They also showed that this involves both emulsification and a chemical cross-linking of protein molecules through disulfide bond formation by sonochemically generated superoxide [31]. Interfacial tension between water and an organic phase was used by Liu et al. to prepare encapsulated assemblies of proteins on organic droplets [32]. These oil/water interfaces while stabilizing the protein at the interface, however have an inherent disadvantage in that the oil droplets are not suitable to dissolve water-soluble guest biopolymers. Morikawa et al. have used ionic liquid–water interface to encapsulate proteins in biopolymers [33]. Here surface-modified protein microcapsules spontaneously form at ambient temperature. The size and rate of formation depend on the type of protein and also on the charge density on the surface of the protein which can be regulated by suitable choice of the anions or cations of the ionic liquid. Following section pertains to use of LbL technique and modification of this technique in the preparation of protein capsules.
2.1. Capsules using covalent interactions 2.1.1. LbL and other modified techniques of LbL Design of microcapsules based on electrostatic interactions has been carried out by layer by layer adsorption (LbL) of polyelectrolytes in many examples including polypeptides built on colloidal templates. This method developed by Decher et al. uses sequential deposition of oppositely charged layers on a template that can then form polyelectrolyte shells [34,35]. Here the shell thickness can be controlled and with suitable triggers can be used to release materials [36,37]. Capsules prepared using the LbL technique have been made using a variety of cargo molecules that include polymers, low molecular weight compounds, and enzymes [38–43]. Zelikin et al. have used a facile method to encapsulate both single as well as double stranded DNA in nanoengineered, degradable polymer
Fig. 1. Scheme showing different methods of preparation for protein microcapsules.
M. Jaganathan et al. / Advances in Colloid and Interface Science 209 (2014) 1–7
Fig. 2. Schematic illustration of fabrication of protein microcapsule via oil/water emulsion using sonication.
microcapsules and have used amine functionalized silica particles as template in which the DNA was adsorbed (Fig. 3.). Then a sequential deposition of thiolated PMA (PMA SH) and poly(vinylpyrrolidone) have been used to form multilayers; followed by cross-linking of the thiol groups of the PMASH in the multilayers into disulfide linkages. Finally the sacrificial SiO + 2 particles have been removed. The DNA has been shown to retain its functionality and structural integrity [44]. In all the capsules formed from the LbL method, the cytotoxicity of synthetic polymer shells often arising from the use of cationic polyelectrolytes seems to be a major disadvantage. De Koker et al. overcame the limitations of toxicity in LbL technique, by using polyelectrolyte capsules consisting of dextran sulfate and polyL-arginine in which antigen proteins were loaded and they showed that such capsules degrade in dendritic cells through proteases and subsequently release the antigen [45]. Temmermann and co-workers [46] evaluated the encapsulation efficiency of such microcapsules and showed that the efficiency is strongly dependent on both the charge and the molecular weight of the protein and on the number of polyelectrolyte bilayers the microcapsules consist of. In the context of use of the microcapsules as carrier for biomedical applications, it is necessary to prepare microcapsules having low toxicity, with robust shells that are stable under extreme acidic and basic conditions, and can withstand high shear force. Ye et al. have used chemically modified silk fibroin to form microcapsules using LbL technique. Here they applied cationic silk-poly- L-lysine (SF-PL) or anionic silk-poly-L-glutamic acid (SF-PG) that allowed the formation of stable
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ultrathin shell microcapsules with a dramatic increase in swelling, thickness, and microroughness at extremely acidic (pH b 2.5) and basic (pH N 11.0) conditions without much disintegration. The additional advantage with these microcapsules is that they undergo remarkable softening with a reduction in Young's modulus by more than 1 order of magnitude due to the swelling, stretching, and increase in material porosity which finds applications in bioengineering [47]. Zhou et al. [48] showed that the poly-L-lysine can be conjugated as a pro-drug of cisplatin (II). Here a Pt (IV) complex with a carboxyl group was synthesized and conjugated to the side chains of poly-L-lysine (PLL), resulting in a polypeptide-drug conjugate, PLL-Pt (IV). PLL-Pt (IV) was then assembled with poly-L-glutamic acid (PGA) through LbL technique on a colloidal support into biodegradable, controllably drugloaded, and environmentally responsive polypeptide capsules. Platinum release from the capsules was found to be greatly enhanced under low pH and reductive conditions. Lee et al. demonstrated the use of LbL designed hyaluronic acid (HA)—poly-L-lysine (PLL) adsorbed systems in which the core removal was carried out by the reducing agent dithiothreitol at neutral pH [49]. The HA/PLL multilayers on the shell were chemically cross-linked via carbodiimide chemistry and showed enhanced physical stability against freeze–thaw cycles and acidic pH conditions. These microcapsules were then used to encapsulate bovine serum albumin (BSA) which could be released using pH gradients or by adding an HA digesting enzyme (hyaluronidase) in the incubation medium.
2.1.2. LbL method combined with post functionalization Improving on the LbL technique Ochs et al. have used an elegant combination of LbL with click chemistry wherein initially poly-L-lysine (PLL) and poly-L-glutamic acid (PGA) have been modified with alkyne (ALK) and azide (AZ) moieties [50]. The capsules obtained for (PLL-AZ/PLL-Alk) multilayer films assembled at pH 5/9 as well as for (PGA-AZ/PGA-ALK) assembled at pH 4 were stable in the range of pH 2 to 11 and exhibited pH-responsive swelling/shrinking. The advantage with this method is that typically individual layers or the capsule surfaces made from biodegradable materials, can be post-functionalized by the use of either excess click groups or other functionality in the film and offer a promising approach towards the design of novel biodegradable vehicles for targeted drug delivery.
Fig. 3. Scheme for encapsulation of DNA in nano-engineered degradable microcapsules.
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Becker and co-workers showed that capsules prepared using LbL technique can be used to deliver small interfering RNAs (siRNA) in prostate cancer cells [51]. They used thiol modified polymethacrylic acid poly-L-lysine (PMASH-PLL) microcapsules. Here a nanoporous particle template was used into which PLL was infiltrated prior to PMASH multilayer assembly. The siRNA cargo was then post loaded into these preformed microcapsules by diffusion through the multilayer film and complexation by the PLL. Correia and co-workers in a recent paper reported on the use of multilayered hierarchical capsules for facilitating cell adhesion sites. Using a simple but elegant method of using liquefied capsules with assembly of poly-L-lysine, alginate, and chitosan they encapsulated surface functionalized poly-L-lactic acid (PLLA) and showed that these can provide support for cellular functions of anchorage dependent cells [52]. The optimal use of bioactivity of protein microcapsules has been demonstrated by Wang et al. and they used the Michael addition and Schiff base reactions in between two layers to form protein microcapsules having a robust but flexible shell [53]. They initiated the process by using a protein-doped CaCO3 template synthesized via co precipitation, which was then coated with a catecholcontaining alginate (AlgDA) layer. The templates were then exposed to ethylenediamine tetraacetic acid disodium (EDTA) solution to dissolve CaCO3. During CaCO3 dissolution, the generated CO2 gas pushes protein molecules moving to the AlgDA layer, and thereby reactions proceed, forming the shell of protein microcapsules. A scheme of the formation of these capsules is presented in Fig. 4. 2.2. Non covalent methods of microcapsule preparation For the first time, Humblet Hua et al. have used protein fibrils to enhance the mechanical strength of the microcapsules. By using different proteins like lysozyme and ovalbumin, they have shown that the variation in the flexibility of the protein fibrils can change the mechanical properties of the capsules. In their experiments they used various ratios of ovalbumin or lysozyme having positive charges with high methoxyl pectin which is anionic and showed that this method does not require any extra salts to be added [54]. Duongthingoc and colleagues using spray dried whey protein close to isoelectric point have shown the formation of an early crust in the microcapsules. Here the protein in the molten globular state creates a cohesive network that has been used to encapsulate yeast cells [55]. Puga and co-workers have demonstrated that pectin-coated chitosan microgels crosslinked on superhydrophobic surfaces can be used to encapsulate 5-fluorouracil which could then be used to deliver the drug onto cancer cells [56]. In the food industry, specifically in the manufacture of nutraceuticals or functional food, the use of spray drying for the production of anhydrobiotics has gained much importance, mainly due to cost efficiencies, enhanced product shelf life. Behboudi-Jobbehdar et al. used a composite of whey protein, maltodextrin and D-glucose carrier to encapsulate the thermo sensitive probiotic lactobacilli strains Lactobacillus acidophilus applying the spray drying technique [57]. The microcapsules obtained were spherical in shape with few surface cavities which seemed to stabilize the lactobacilli.
Mao et al. in their work have demonstrated elegantly the use of mixed protein systems as emulsions to deliver carotenoids [58]. In this work, oil in water emulsions of β-lactoglobulin, lactoferrin and their mixed systems have been analyzed for their suitability to stabilize and deliver β-carotene. The method seems to suggest a simple delivery vehicle for bioactive components. Matalanis and McClements have demonstrated the use of hydrogel microspheres fabricated from a phase separated mixture of pectin, caseinate, and emulsified oil to form an oil-in-water-in-water (O/W1/W2) emulsion which has been then acidified and finally cross-linked with transglutaminase. Such microspheres trapped within bioactive molecules have been used efficiently to deliver lipophilic agents. Using various proportions of the continuous biopolymer phase, the authors could optimize the yield as well as the performance of the microspheres [59]. Chen and co-workers used whey protein isolate and sodium caseinate to encapsulate lipophilic bioactive components like fish oil, phytosterols and limonene and preserve the flavor and odor. The thickness of the protein walls of the capsules could be regulated by the ratio of the whey protein to the caseinate [60]. In the delivery of DNA for therapeutics as well as in studies on hybridization often carriers are used that can deliver the nucleotides specifically. Hardin et al. used gelatin microcapsules which were initially fabricated using microfluidics [61]. Microcapsules were loaded with the 15 base long secondary DNA targets, then capped with a polyelectrolyte bilayer and finally with a monolayer of polystyrene microcapsules. When the capsules were warmed to 37 °C, secondary DNA targets were released from the gelatin template and then displaced the shorter, original hybridization partners on the polystyrene microcapsules. Mazzitelli et al. in a two-step gelation process have used pectin and gelatin [62]. Here, the first thermal step helps in gelation of gelatin followed by an ionic one where gel structure of pectin occurs. The viscoelastic properties of these gel combinations have been optimized to make an efficient transdermal patch. These patches have been used as transdermal system to deliver testosterone and the in vitro drug release profiles have been analyzed. Shimokawa et al. have used gelatin– water–lower alcohol systems in different proportions to form microcapsules by coacervation method and have analyzed the drug release profile from the different ternary systems [63]. Normally in the development of drugs using peptides and proteins the difficulty lies in their slow clearance in the liver and other body tissues by proteolytic enzymes. Hence there is a difficulty to administer these drugs through regular injections. Zheng et al. have designed and applied a biodegradable and redoxresponsive submicron capsules using the LbL technique with poly-Laspartic acid and chitosan for transmucosal delivery [64]. Erokhina and co-workers have recently reported on a novel method of using collagen type I microcapsules and the release mechanism from these capsules has been carried out using a biological stimulus from an over-expressed collagenolytic matrix metalloproteinase I (MMPI) [65]. This method requires no external labels to study the release kinetics. Here the release of any drug through specific pore opening on the microcapsule surface can be regulated. Thus, this allows control of the release time and a variety of drugs of different sizes can be released.
Fig. 4. Scheme for protein capsules through template mediated interfacial reaction.
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Most of the methods described so far are based on some form of chemical microencapsulation methods. Most application scientists however associate the chemical methods with strong operating conditions and feel it suffers from non-green technologies. 2.3. Protein microcapsules using ‘freeze–thaw’ technique Among the noncovalent methods for preparing the protein microcapsules already mentioned above, most methods use amphiphilicity of the molecules for effecting hydrophobic interactions or even to create an interface. However Daamen et al. in 2007 reported for the first time the use of a simple method to prepare biocapsules at ambient or low temperatures from a wide class of biomolecules. This method is not dependent on the amphiphilicity of the biomolecules and uses a combination of freezing, annealing, and lyophilization procedure [66]. Microcapsules of extra cellular matrix protein elastin which is fibrous, in the size range 200 nm to 10 μ, have been designed using a soluble form of the protein in the above protocol. Microcapsules of a variety of macromolecules, including serum albumin, a globular protein, type I collagen (280 kDa) a fibrous protein and heparin as an example of a highly negatively charged polysaccharide have been prepared by this method. Using this technique, they demonstrated that this method can encapsulate both hydrophilic and lipophilic compounds, and can be prepared in large quantities. They described the mechanism of capsule formation by suggesting a three step process of microphase separation by fast-freezing, structural rearrangement by annealing to room temperature followed by the creation of a lumen by lyophilization. In their work, they had suggested the use of droplets of the protein which can be fast-frozen in liquid nitrogen and capsules prepared in high quantities. Using this droplet technique, Daisy Rani et al. prepared microcapsules of fibrinogen, a fibrous protein which was used as template to form nano clusters of nickel oxide [67]. Here the capsules of complexes of nickel–fibrinogen formed stable coatings on solid substrates which were then subjected to heating to form nickel hydroxide and subsequently nickel oxide. Madhumitha and Dhathathreyan have prepared microcapsules of bovine serum albumin (BSA) in the size range 0.2 to 0.3 μ. High resolution transmission electron micrograph of the capsules prepared at pH = 7.5 is shown in Fig. 5 [68]. The samples for TEM shown in the study do not have any external staining agent. In this technique capsules of the protein at pH 7.5 were prepared by dissolving the protein in phosphate buffer and introduced as droplets in a container with liquid nitrogen which was then slowly brought to − 20 °C for 2 h (annealing step) after which it was slowly brought to 5 °C and then crosslinked with 0.1% glutaraldehyde. Solutions of
Fig. 5. High resolution TEM of bovine serum albumin capsules at pH = 7.5.
Fig. 6. Scheme for preparation of protein microcapsules using the ‘freeze–thaw’ technique.
the protein at different pH (pH ranging from 3.5 to 8.5) were used in the above method and stable capsules of the protein at all pH could be obtained. A scheme for this method is shown in Fig. 6. Maheshkumar and Dhathathreyan used the ‘droplet technique’ and designed hemoglobin capsules in the size range of 0.1 to 0.3 μ which were then stabilized as films at air/buffer and solid/air interface [69]. After the freeze–thaw cycle they were then crosslinked with 0.1% glutaraldehyde. Particle size analysis of the capsules carried out using dynamic light scattering showed a major population (69%) with an average size of about 0.1–0.2 μ and populations of 1 to 2 μ (18%) and the rest in the range 3–4 μ. Fig. 7(a) and (b) shows the transmission electron micrographs of the capsules at pH 5.5 and 7.5 respectively. The samples formed were spread as films at the interface by spreading them on the buffer of pH 6.8 (pI of the protein). This method of stabilizing different protein films by spreading them on buffers at the isoelectric point has been used by Dhathathreyan and co-workers [70–72]. The average surface area versus surface pressure isotherms for these spread films showed stable films at the air/buffer interface. The dilational rheology of the capsules of the protein spread in a Langmuir was studied using oscillatory compressions of the film and by analyzing the surface pressure and the phase shift between the surface tension signal and the surface area. The protein capsules are composed of both hydrophobic and hydrophilic segments in appropriate proportion, and at the air/water interface they exhibit properties of a Janus-type particle with hydrophilic groups oriented towards the bulk water and the hydrophobic moieties organizing towards the air interface. The local interactions due to the asymmetry experienced by the water molecules at the protein/water interface and the changes in hydrophobic interactions seem to drive the assembly process. Due to strong stabilizing effect of the lateral electrostatic repulsions, such monolayer when compressed forms close-packed hexagonal structure, and can be easily transferred onto a solid substrate with the Langmuir–Blodgett technique. The results from their work suggest that stable capsules of pure proteins can be designed as films that can find biomedical applications and can be used to encapsulate small peptides and some lipophilic compounds. The micrographs in Fig. 7 suggest that this technique results in multicore microcapsules which can then be used either to diffuse or dissolve the contents and then release them. The model of formation of the protein capsules is complex and involves many processes such as capsule–interface interactions, protein orientations on the surface, lateral protein–protein interactions, bound water to free water ratios on the surface. Further secondary structural analysis and mass spectroscopy showed that the capsules had hemoglobin in the native state in tetrameric form suggesting that half life can be increased and corresponding toxicity also can be controlled. Duan et al. reported a simple and controllable method of using a CaCO3 particle as template to design Hb spheres with a high loading content in combination with the LbL technique [73]. The surface of the
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Fig. 7. Transmission electron micrographs of hemoglobin at (a) pH = 5.5; (b) pH = 7.5.
Hb spheres was then chemically modified by biocompatible polyethylene glycol to protect and stabilize the system. Using the freeze–thaw technique, Maheshkumar and Dhathathreyan have used the microcapsules of Cytochrome c (cyt c) prepared to model fibril formation. Fig 8(a) shows the TEM micrograph of the protein capsules and Fig. 8(b) shows the particle size analysis of the capsules. The capsules of cytochrome c obtained were subjected to repeated compression and expansion at air/buffer interface and the microcapsules are elastic up to some pressure beyond which they start to fuse. Till the point of fusion the conformation of the protein in the capsules remains fairly unchanged. However on fusion, the protein seems to undergo dramatic change in the secondary structure and transits to a more beta sheet like structure from the original helical structure.
3. Conclusion In summary, several techniques exist for preparation of robust assembly of protein microcapsule using different driving forces like amphiphilicity, electrostatic interactions, hydrogen bonding and hydrophobic interactions at fluid–fluid interfaces using both covalent and non covalent interactions between the biopolymer and either the active matter or any other functionality. The non covalent method seems to result in microcapsules with multicore structures for the proteins. The surface of the protein microcapsules can be easily modified by functional materials such as quantum dots and these can be conjugated with other molecules. By using the same strategy, numerous functional materials can be displayed on the protein microcapsules for a variety of biotechnological and biomedical applications. Significant strides have been made in the design and application of proteinaceous materials in microcapsules as carriers for food, nutraceuticals, dyes and for drug encapsulation. Improving the stability, sizes and overall properties of the capsules for immobilizing bioactive molecules is of great interest and more research needs to be carried out in this direction. Acknowledgments One of the authors MJ would like to thank DST-INSPIRE for fellowship. The authors would like to thank the Council of Scientific and Industrial Research (12th plan supra-institutional project-STRAIT-A/2013/BPY/ CSC0201/1035) for funding. References [1] [2] [3] [4]
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