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A rapid increase in anthropogenic emission of greenhouse gases, mainly carbon dioxide, has been a growing cause for concern. While photocatalytic reduction of carbon dioxide (CO2) into solar fuels can provide a solution, lack of insight into energetic pathways governing photocatalysis has impeded study. Here, we utilize measurements of electronic density of states (DOS), using scanning tunneling microscopy/spectroscopy (STM/STS), to identify energy levels responsible for photocatalytic reduction of CO2-water in an artificial photosynthetic process. We introduce desired states in titanium dioxide (TiO2) nanoparticles, using metal dopants or semiconductor nanocrystals, and the designed catalysts were used for selective reduction of CO2 into hydrocarbons, alcohols, and aldehydes. Using a simple model, we provide insights into the photophysics governing this multielectron reduction and design a new composite photocatalyst based on overlapping energy states of TiO2 and copper indium sulfide (CIS) nanocrystals. These nanoparticles demonstrate the highest selectivity for ethane (>70%) and a higher efficiency of converting ultraviolet radiation into fuels (4.3%) using concentrated sunlight (>4 Sun illumination), compared with platinum-doped TiO2 nanoparticles (2.1%), and utilize hot electrons to tune the solar fuel from alkanes to aldehydes. These results can have important implications for the development of new inexpensive photocatalysts with tuned activity and selectivity.

The potential of cyclodextrins to stabilize alpha-chymotrypsin upon encapsulation in Poly(lactic-co-glycolic) acid (PLGA) microspheres using a solid-in-oil-in-water (s/o/w) technique was investigated. Two cyclodextrins, hydroxyl-propyl-beta-cyclodextrin (HPbetaCD) and methyl-beta-cyclodextrin (MbetaCD), one insoluble and the other soluble in methylene chloride, were used. The results demonstrate that HPbetaCD failed to stabilize alpha-chymotrypsin upon encapsulation. Specifically, 19% of the protein was aggregated and the specific activity of the enzyme was reduced to ca. 50% of that prior to encapsulation. In contrast, MbetaCD significantly decreased the formation of aggregates to 3% and the retained specific activity of the enzyme was approximately 90%. The co-lyophilization of alpha-chymotrypsin with MbetaCD prior to encapsulation was a requisite to preserve the protein stability in microspheres. Furthermore, MbetaCD prevented the loss of protein during the preparation of microspheres and the encapsulation efficiency was improved to 90%. Release experiments showed the use of MbetaCD modified the release profile: the burst release decreased from 54% (in the absence of the excipient) to 36%. The results suggest that MbetaCD might be a suitable excipient to improve protein stability in s/o/w encapsulation procedures.

Poly(ethylene glycol) (PEG) was used as emulsifier to prepare alpha-chymotrypsin-loaded poly(lactic-co-glycolic) acid (PLGA) microspheres by a solid-in-oil-in-water (s/o/w) technique. The effect of the molecular weight of PEG on protein stability was assessed by the determination of the amount of insoluble aggregates, the activity loss and the magnitude of structural perturbations. In addition, the effect of the molecular weight of PEG on the encapsulation efficiency, microsphere characteristics and release kinetics was investigated. X-ray photoelectron spectroscopy (XPS) was employed to characterize the surface chemistry of the microspheres. Microspheres were prepared using PEG with molecular weight of 6,000, 8,000, 10,000, 12,000 and 20,000. The results indicate that PEG 20,000 was the most effective emulsifier when producing alpha-chymotrypsin-loaded microspheres with respect to protein stability. The aggregate formation was decreased from 18% to 3%; the protein inactivation and the encapsulation-induced structural perturbations were largely prevented. XPS confirmed that PEG was largely located on the surface of microspheres. The molecular weight of PEG affected the microspheres' characteristics and release kinetics. Microspheres prepared with PEG 20,000 showed improved encapsulation efficiency (80%) and a continuous release (for 50 days) with the lowest amount of initial release. It is demonstrated that the selection of the optimum molecular weight of PEG when used as emulsifier in the preparation of microspheres is a critical factor in the development of sustained-release formulations for the delivery of proteins.

The effectiveness of the covalent modification of alpha-chymotrypsin with methoxy poly(ethylene glycol) (PEG) to afford its stabilization during encapsulation in poly(lactic-co-glycolic) acid (PLGA) microspheres by a solid-in-oil-in-water method was investigated. alpha-Chymotrypsin was chemically modified with PEG (M(w) = 5000) using molar ratios of PEG-to-chymotrypsin ranging from 0.4 to 96. Various conjugates were obtained and the amount of PEG modification was determined by capillary electrophoresis. In this investigation, only those conjugates with PEG/chymotrypsin molar ratios between approximately 1 and 8 were considered because higher levels of modification caused protein instability even before encapsulation. The stability and functionality of the chymotrypsin formulations were investigated before encapsulation by measuring enzyme kinetics, thermal stability, and tertiary structure intactness, and after the initial lyophilization process by determining the secondary structure content. These stability parameters were related to select ones after encapsulation in PLGA microspheres (specifically, the amount of insoluble aggregates, residual enzyme activity, and magnitude of protein structural perturbations). The results show that the more stable the protein conformation before encapsulation was, the higher was the retention of the specific activity after encapsulation. In contrast, no relationship was found between the protein stability before encapsulation and the magnitude of encapsulation-induced protein aggregation. Even the lowest level of modification (PEG-to-chymotrypsin molar ratio of 0.7) drastically reduced the amount of insoluble aggregates from 18% for the nonmodified protein to 4%. The results demonstrate that PEG modification was able to largely prevent chymotrypsin aggregation and activity loss upon solid-in-oil-in-water encapsulation in PLGA microspheres. It is demonstrated that it is essential to optimize the degree of protein modification to ascertain protein stability upon encapsulation., (Copyright 2004 Wiley-Liss, Inc.)

Purpose: To investigate the potential of different solvents with better biocompatibility to replace CH2Cl2 in the encapsulation of alpha-chymotrypsin in poly (lactic-co-glycolic) acid (PLGA) microspheres without causing protein instability., Methods: The oil-to-water (O:W) ratio in the emulsification step of the solid-in-oil-in-water (s/o/w) encapsulation process was optimized with respect to maximizing protein stability and encapsulation efficiency for various solvents. Formation of insoluble aggregates and residual enzyme activity were primarily used as stability parameters. Several solvents possessing low toxicity with different water solubility were used to prepare alpha-chymotrypsin loaded PLGA microspheres., Results: The O:W ratio in the emulsification step is critical with respect to maintaining protein stability. This was related to the solvents' water solubility. In general, hydrophilic solvents were detrimental to protein stability and encapsulation efficiency. However, after optimization of the O:W ratio for solvents with different water solubility, protein stability was preserved during encapsulation using butyl acetate when poly (ethylene glycol) (PEG) was used as the emulsifying agent (ca. 1% of non-covalent aggregates and 93 +/- 10% of residual specific activity)., Conclusions: The s/o/w technique was successfully improved by replacing the ICH class 2 solvent CH2Cl2 with the class 3 solvent butyl acetate without compromising alpha-chymotrypsin stability.

Protein aggregation and inactivation are major problems associated with the encapsulation of pharmaceutical proteins in biodegradable microspheres. The objectives of this study were to identify the causes of aggregation and inactivation of two model enzymes upon solid-in-oil-in-water (s/o/w) encapsulation in poly(lactic-co-glycolic) acid (PLGA) microspheres in order to rationally develop approaches assuring their stability. S/o/w encapsulation of gamma-chymotrypsin in PLGA microspheres caused aggregation of ca. 30% and halved its specific activity. Co-lyophilization with poly(ethylene glycol) (PEG) substantially reduced the loss in enzyme activity but 8% of the protein still aggregated during encapsulation. Model studies performed under conditions relevant to the encapsulation procedure allowed pinpointing the cause of gamma-chymotrypsin instability, which was mainly the formation of the oil-in-water emulsion. To prevent aggregation in this encapsulation step, the most commonly used emulsifying agent polyvinyl alcohol (PVA) was replaced by PEG because it is known to reduce protein aggregation at interfaces. The use of PEG as the emulsifying agent in the aqueous and organic phase prevented gamma-chymotrypsin inactivation and aggregation during encapsulation. The stabilization approach also worked for the model protein horseradish peroxidase and thus is of a general nature.

Development of alternative procedures to the commonly employed water-in-oil-in-water technique to encapsulate proteins in polymers is needed due to protein stability issues. Herein the model protein gamma-chymotrypsin has been encapsulated in poly(D,L-lactic-co-glycolic)acid (PLGA) microspheres using the solid-in-oil-in-water (s/o/w) encapsulation technique. The model protein was chosen because it has a measurable biological activity and its unfolding is irreversible. The latter make the protein an excellent sensor for unfolding events in the encapsulation procedure. While lyophilization did not cause any irreversible aggregation or loss in activity, encapsulation of the lyophilized enzyme by the s/o/w technique proved detrimental to its integrity. Specifically, 34% of the encapsulated protein was aggregated and the specific activity of enzyme released within 24 h was reduced to ca. 50% of that prior to encapsulation. FTIR spectra demonstrated substantial encapsulation-induced perturbations of the secondary structure of gamma-chymotrypsin. To achieve stabilization of gamma-chymotrypsin during encapsulation, excipients were employed during the initial lyophilization process. When gamma-chymotrypsin was co-lyophilized with poly(ethylene glycol) (PEG) the formation of non-covalent aggregates inside the microspheres decreased significantly to 8%. FTIR data showed that PEG prevented encapsulation-induced structural perturbations. In contrast, the amount of aggregates remained high (34%) when gamma-chymotrypsin was co-lyophilized with trehalose. No additional non-soluble aggregates were formed during 1 week of in vitro release. Furthermore, the amount of non-soluble aggregates in the microspheres after encapsulation correlated with the amount of non-released protein. Therefore in vitro release did not cause aggregation. Similar results were found with respect to the retention of the specific enzyme activity where PEG afforded excellent stability.

Sustained release of pharmaceutical proteins from biocompatible polymers offers new opportunities in the treatment and prevention of disease. The manufacturing of such sustained-release dosage forms, and also the release from them, can impose substantial stresses on the chemical integrity and native, three-dimensional structure of proteins. Recently, novel strategies have been developed towards elucidation and amelioration of these stresses. Non-invasive technologies have been implemented to investigate the complex destabilization pathways that can occur. Such insights allow for rational approaches to protect proteins upon encapsulation and release from bioerodible systems. Stabilization of proteins when utilizing the most commonly employed procedure, the water-in-oil-in-water (w/o/w) double emulsion technique, requires approaches that are based mainly on either increasing the thermodynamic stability of the protein or preventing contact of the protein with the destabilizing agent (e.g. the water/oil interface) by use of various additives. However, protein stability is still often problematic when using the w/o/w technique, and thus alternative methods have become increasingly popular. These methods, such as the solid-in-oil-in-oil (s/o/o) and solid-in-oil-in-water (s/o/w) techniques, are based on the suspension of dry protein powders in an anhydrous organic solvent. It has become apparent that protein structure in the organic phase is stabilized because the protein is "rigidified" and therefore unfolding and large protein structural perturbations are kinetically prohibited. This review focuses on strategies leading to the stabilization of protein structure when employing these different encapsulation procedures.

Bovine serum albumin (BSA) was encapsulated into poly(lactide-co-glycolide) (PLG) microspheres by a solid-in-oil-in-water (s/o/w) technique. We tested whether perturbations in BSA secondary structure could be minimized during encapsulation by using trehalose and how this would influence BSA aggregation and release. BSA secondary structure was monitored noninvasively by Fourier-transform infrared spectroscopy. When BSA was co-lyophilized with trehalose, lyophilization-induced structural perturbations were significantly reduced. The formulation obtained (BSA-Tre) was encapsulated into PLG microspheres and, by optimizing critical encapsulation parameters, a loading efficiency of 85% was achieved. However, due to the loss of the excipient in the o/w emulsion step, the structure of BSA-Tre was more perturbed than before encapsulation. Excipient-loss and encapsulation-induced structural perturbations could be prevented by saturating the aqueous phase in the o/w step with trehalose and by using the organic solvent chloroform. This in turn reduced the formation of soluble BSA aggregates. BSA was released from PLG microspheres using the improved formulations with an initial release in 24 h of not more than 22%, followed by a sustained release over at least 2 weeks. In summary, optimization of the encapsulation conditions in the s/o/w procedure resulted in the encapsulation of BSA without procedure-induced structural perturbations and minimized the release of aggregated protein. This demonstrates that the s/o/w technique is an excellent alternative to the most common encapsulation procedure, namely the water-in-oil-in-water technique.

Non-aqueous protocols to encapsulate pharmaceutical proteins into biocompatible polymers have gained much attention because they allow for the minimization of procedure-induced protein structural perturbations. The aim of this study was to determine if these advantages could be extended to a semi-aqueous encapsulation procedure, namely the solid-in-oil-in-water (s/o/w) technique. The model protein bovine serum albumin (BSA) was encapsulated into poly(lactide-co-glycolide) (PLG) microspheres by first suspending lyophilized BSA in methylene chloride containing PLG, followed by emulsification in a 1% aqueous solution of poly(vinyl alcohol). By variation of critical encapsulation parameters (homogenization intensity, BSA:PLG ratio, emulsifier concentration, ratio of organic to aqueous phase) an encapsulation efficiency of > 90% was achieved. The microspheres obtained showed an initial burst release of < 20%, a sustained release over a period of about 19 days, and a cumulative release of at least 90% of the encapsulated BSA. Different release profiles were observed when using different encapsulation protocols. These differences were related to differences in the microsphere erosion observed using scanning electron microscopy. Release of BSA was mainly due to simple diffusion or to both diffusion and microsphere erosion. Fourier-transform infrared studies were conducted to investigate the secondary structure of BSA during the encapsulation. Quantification of the alpha-helix and beta-sheet content as well as of overall structural changes showed that the secondary structure of encapsulated BSA was not more perturbed than in the lyophilized powder used initially. Thus, the encapsulation procedure did not cause detrimental structural perturbations in BSA. In summary, the results demonstrate that the s/o/w technique is an excellent alternative to the water-in-oil-in-water technique, which is still mainly used in the encapsulation of proteins in PLG microspheres.