Osmosis-Driven Protein Distribution Optimization

Osmosis-Driven Protein Distribution Optimization Osmosis-Driven Protein Distribution Optimization via a Single-Vessel Process Purpose Protein-loaded microspheres prepared via the water-in-oil-in-water (w/o/w) emulsion method are porous with poor protein distribution due to the inner water phase leaving the microspheres following polymer precipitation, resulting in the generation of cavities and large pores. Attempts to prevent this by osmosis-driven escape of the inner water phase were unsuccessful, as a polymer precipitation front formed instantly resulting in hollow microspheres. A method to tightly control the events during microsphere formation was developed. Polymer precipitation was controlled by saturating the outer water phase in the  organic solvent and isolating the secondary emulsification step from the environment to prevent organic solvent extraction prior to osmosis-driven water escape. Moreover, a single-vessel microsphere preparation method was developed to eliminate product loss and contamination resulting from multiple vessel transfers. Methods Microspheres were prepared in a single vessel, from polymer dissolution to final product storage (fig. 1). BSA-FITC, 25 kDa PLGA 50:50 and methylene chloride (DCM) were used. 0.1% w/v PVA in water was used as the outer water phase, with or without 1% w/v NaCl. The microspheres were characterized by SEM, confocal microscopy, drug loading, encapsulation efficiency, and burst release (24h) at 37oC. Results SEM and confocal microscopy revealed non-porous microspheres with homogenous distribution of protein throughout the polymer matrix when osmosis was applied, as opposed to microspheres where no osmosis was applied (Figure 2). Applying osmosis without isolating the system in a closed vial resulted in hollow microspheres. Moreover, while the application of osmosis in the preparation process did not alter significantly the drug loading or the encapsulation efficiency, the 24 h burst release decreased dramatically as shown in Figure 3. Conclusion Our results confirmed our hypothesis that isolating the system in a closed vial during the second emulsification step in combination with application of osmosis prevents early DCM extraction and polymer precipitation, resulting in non-porous microspheres with homogenous protein distribution. Protein-loaded microspheres prepared via this method showed low burst release, a significant property for controlled release microsphere systems. This work showcases the importance of excipients during microsphere preparation, as the careful choice of excipients significantly affects product quality and performance. Opalescence and Liquid-Liquid Phase Separation in a Protein Solution Purpose To study the effect of concentration, temperature, pH, ionic strength and buffer species on opalescence and liquid-liquid phase separation (LLPS) in a protein solution Methods Turbidity of a protein solution was measured as percent transmittance using UV-Vis spectrophotometer at different solution conditions. LLPS of the protein was confirmed by equilibrium studies and by visually observing LLPS under microscope. Structural changes in protein before and after phase separation were studied using Circular Dichroism. Temperature ramp studies were conducted to determine the phase separation temperature (Tcloud) at different solution conditions. Tcloud (temperature where transmittance is 70%) was correlated to protein-protein interactions measured in dilute solutions using DLS (kD). Opalescence measurements, Tcloud measurements and DLS studies were also performed in the presence of different salt ions at pH 6.5 and 15 mM ionic strength. Results For the protein molecule studied maximum opalescence is observed near the pI of the molecule and at low ionic strengths. From equilibrium studies, it was observed that protein concentration remains constant in two phases (protein-rich and protein-poor) irrespective of the initial protein concentrations. At low ionic strength and close to the pI of the molecule, Tcloud values were higher indicating a relatively less stable solution, which shifted to lower temperature values at pH away from pI. At relatively higher ionic strength, Tcloud decreased at a pH closer to pI of the molecule and increased away from pI. There was discrepancy in the magnitude of the change in attractive interactions measured by DLS and shift in Tcloud with change in solution conditions. Conclusion This study shows that phase separation is an equilibrium/thermodynamic process; protein does not undergo structural changes on phase separation. Strong attractive interactions are observed in systems exhibiting LLPS as indicated by shifts to higher Tcloud and large negative kD. Tcloud measurements can be utilized as a potential screening tool to study the effect of excipients on opalescence and phase separation in early stages of protein formulation development. A Detailed Mechanistic Study on Adjuvants and Optimizing Antigenicity of Particulate Cancer Vaccines Purpose Sperm protein 17 (Sp17) is a cancer/testis antigen which is expressed aberrantly in several cancers like prostate, ovarian cancer, hepatocellular carcinoma and multiple myeloma. Its restricted expression in normal tissues and aberrant expression in cancers makes it an attractive target for cancer immunotherapy. Enhancing delivery of Sp17 may significantly improve clinical outcome by eliciting a specific and sustained anti-tumor response. The main goals of this project were 1) to formulate Sp17 microparticles (MP) and investigate its efficacy in vitro. 2) Conduct a detailed mechanistic study on adjuvants that may augment anti-tumor efficacy of Sp17 MP. 3) To test combination of two adjuvants in conjunction with Sp17 MP for synergetic effect Methods Recombinant Sp17 was expressed in M15 cells, isolated and purified using the Ni-NTA fast start kit (Qiagen). Sp17 and adjuvants were encapsulated separately in MP using the Buchi B-290 spray drier. Particle size, zeta potential and SEM imaging was performed on microparticles. SDS-PAGE was performed to confirm the stability of Sp17 in MP. Release of Sp17 from MP was performed in gastric and intestinal pH conditions. Eight adjuvant MP were screened on DC 2.4 cells by studying several innate and adaptive immune markers like nitric oxide, CD40, CD80, CD86, CD54 and MHC-II. Results Sp17 MP had an average particle size of 3.59  ± 0.5ÃŽ ¼m and zeta potential of +9.36mV. Encapsulation efficiency of Sp17 was found to be 78%. SEM images confirmed particles were irregular in shaped with surface indentations. SDS-PAGE confirmed the presence of Sp17 encapsulated in its native form. Cumulative release of Sp17 was approximately 15% in simulated murine gastric and intestinal pH conditions. Nitric oxide release was significantly (p compared to Sp17 MP. Combination of R848 and Alum, R848 and MF59 and R848 and P4 showed enhanced expression of CD80. CD40 elevation was highest in MPL and R848 combination. Conclusion Sp17 MP in combination with R848, MPL and MF59 MP significantly improve innate and adaptive immune response to cancer antigens. In Vitro and In Vivo Studies on Transdermal Particulate HPV Vaccine Purpose Human Papillomavirus (HPV) vaccines are recommended by the World Health Organization for cervical cancer control programs world-wide. However, the cost of these vaccines and requirements for administration are significant barriers for vaccination in developing countries. Microparticulate vaccines have the potential to alleviate these problems. The purpose of this study is to develop an HPV16 microparticulate vaccine for transdermal administration and evaluate its efficacy in both in-vitro and in-vivo studies. Methods HPV 16 virus-like particles (VLPs) were produced in human embryonic kidney cells 293TT. VLPs were incorporated into a cellulosic polymer matrix and formulated into microparticles using a Buchi B-290 spray dryer in a single step. VLP encapsulation was determined using transmission electron microscopy (TEM) and Western blot analysis. For in–vitro study, antigen-presenting cells (APCs) were exposed to vaccine and characterized for cell-surface expression (CD40, CD80/86 and MHC II). For in-vivo study, AdminPatch ® transdermal administration of VLPs as microparticles was compared to VLPs in solution. Female BALB/c mice (n=6 for each group) received 4 doses. Blood samples were collected and antibodies were detected with a direct HPV16 VLP IgG ELISA. Spleen and lymph node pools were prepared at week 40 to analyze memory T and B cells using flow cytometry. Results The microparticle yield after spraying was 90% w/w, with average size 3.5+ 0.6 ÃŽ ¼m and average zeta potential -19.7 + 0.3 mV. VLP encapsulation efficiency was 85% based on Western blot detection of HPV16 L1 protein. APCs expressed significantly higher CD40, CD80/86 and MHCII in the particulate vaccine group compared to the solution group. HPV 16 antibodies were detected more frequently in the microparticle group (3 of 6 mice by week 7 and 6 of 6 mice by week 12) than in the solution group (1 of 6 mice by week 12). Spleen and lymph node CD4+, CD27, CD62L and CD45R cell populations were significantly higher (p Conclusion Transdermal administration of HPV VLP as microparticulate vaccine is more immunogenic than HPV VLP in solution. Phase Separation and Component Crystallization in Freezing Segment of Protein and Amino Acid Lyophilization Purpose Many freeze-dried protein formulations contain glass-forming stabilizing excipients (e.g., trehalose) that protect proteins from dehydration-induced irreversible conformation changes and chemical changes during storage. Some amino acid excipients also form glass-state solids upon lyophilization. The purpose of this study was to elucidate miscibility of proteins and amino acid excipients in frozen solutions and its effect on their crystallization. Methods Aliquots of frozen solutions containing a model protein (e.g., recombinant human albumin) and amino acids were applied for heating thermal analysis from -70 °C to obtain glass transition temperatures of maximally freeze-concentrated solutes Tgà ´Ã¢â€š ¬Ã¢â€š ¬Ã¢â€š ¬OEand solute crystallization peaks. Some frozen solutions were annealed at elevated temperatures (e.g., -10 °C) before their second scan from -70 °C. Results Some amino acid excipients (e.g., L-valine, glycine) showed high propensity to crystallize during the freezing process. Other excipients freeze-concentrated into narrow non-ice regions between ice crystals remained amorphous (e.g., sodium Lglutamate, L-arginine hydrochloride) or crystallized (e.g., L-histidine hydrochloride) upon the annealing. Frozen solutions containing the protein and amorphous excipients showed single or double Tgà ´Ã¢â€š ¬Ã¢â€š ¬Ã¢â€š ¬OE transitions that indicate their varied miscibility depending on the combinations and concentration ratios. Many protein-rich frozen solutions showed single Tgà ´Ã¢â€š ¬Ã¢â€š ¬Ã¢â€š ¬OE transitions in the first heating scans and after their annealing, indicating maintenance of the amorphous concentrated solute mixture. Frozen solutions containing rHA and higher mass ratio of L-Arg HCl showed double Tgà ´Ã¢â€š ¬Ã¢â€š ¬Ã¢â€š ¬OE transitions. The transition temperature profiles suggested separation of the non-crystalline solutes into the solute-mixture and excipient phases. Frozen solutions containing rHA and higher mass ratio of L-His HCl showed the amorphous/amorphous phase separation and following crystallization of the excipient. Conclusion The phase separation should allow nucleation of amino acid crystals in the excipient-dominant concentrated phase. Information on the solute mixing state should be valuable for appropriate use of the amino acid excipients either as a crystalline bulking agent or an amorphous stabilizer in freeze-dried formulations.

Auto biography of a Tree Essay

I am a tree, tall and imposing, standing all alone near the campus of a temple. My life is ever so interesting as, I see a huge spectrum of society, funny gestures of people, and hear all kinds of conversation of the devotees who pass by me. I was not always so huge. As is true of all living beings I was also a long time back, a young sapling with this huge tree within me. At that time I looked fresh and beautiful as all other beings who are young, but, at that time I was of course not so imposing. This is what I have seen about life that, every stage has something good about it. I understand that, I am a peepal tree which just grows anywhere and everywhere. Ever since I was young, I remember several people coming to my root and worshipping me. They would light an earthenware lamp, and put it near my root, say their prayers and go away. This was a daily ritual which scores of people followed at my root ever since I can remember. As I started growing up bigger and bigger, the temple authorities put a two feet wall like boundary around my trunk. This was done to protect me from being destroyed by crowds who thronged to me. Since I am near a temple, I have always had the pleasure of a lot of company daily and, the great honour of being worshipped by scores of believers every day. Now, I am a full grown tree, and that two feet wall has been converted into a broad platform all around me, with my root out of sight, and the trunk also somewhat covered from vision. Here on the platform people sit and say their prayers and relax. At times they also consume their temple prasad while sitting on this platform. Oh, what a wonderful feeling it is to be so loved and cared for and, above all, being so honoured and respected. This honour is given to me as, I understand now that, a section of the Indian society considered me a holy tree to be worshipped. This is why there is so much hype about me and my kin. As I stand here, near a temple, I never ever feel lonely I get all sorts of company throughout the day, so, where is the scope of feeling lonely. Early in the morning the temple is opened at 6 a.m. It is cleaned and washed so, I get the company of people who clean the premises. They come to me also and broom the area around me, wash the platform and I am trim and neat to welcome my guests for the day. After the temple is cleaned there is a pooja, and from 7 a.m. devotees start pouring in, and the temple bells start ringing and breaking the silence of the night. Devotees continue pouring in the temple right from 7 a.m. to 10 p.m., when the temple closes. These long hours of the day, there is no chance of my getting bored as, I have the company of so many people moving around the area. Even though I am alone, a lonely tree but, my life is full of thrill and excitement, as I get lots of, and variety of company. This in turn is an added honour for me that, while my friends have the company of trees only, I have the company of human beings who talk and walk and discuss matters to make my life more lively and enjoyable. I often wondered to myself as to why there is so much of greatness thrust upon me, though I am just a tree like any other. Yes, here is the catch, I believe I am so revered because I am considered by the Hindus, a religious sect as a holy tree, an incarnation of their God. Aha! this makes me feel proud of myself and I am prone to thank God for this birth in which I get honour, respect, love and care. What else could any living being aspire for. I and my honour are further enhanced as, I stand near a temple of God. Now, all my anxiety is put to rest as, I have learnt that I am also here to be worshipped just as God himself. I stand here alone yet in great company of human beings. My life is just wonderful, I pray to God that HE grant such a wonderful and eventful life to all humans. One thing I forgot to tell you all, about my life. When people come and sit on the platform around me, I also get a lot of information about man’s world. I hear scandals, I hear about murders, thefts and what all evils that exist in this man’s world. At times I also get to hear small children saying lovely words to their mothers and, seeing the mothers cajoling them, I feel what a beautiful life men have. Thus to add to my experience of life, I have come to know a lot about human beings and their lives. Their lives are also full of pleasures and disappointments. It is not that men only enjoy as, most of us lesser beings seem to feel. Men also have their own problems. So, to add up I’d say my life is a pleasure and a rich experience.

What is Automobility?

Automobility is a concept based on two fundamental ideas.   Autonomy is the self-directing or self-governing freedom of an individual.   Mobility is the movement or travel of an individual.Therefore, automobility is the independent, self-governing movement of an individual.   The concept sounds valid and many people would believe it to be so, however, after a more detailed look automobility may not be as mobile or self-regulating as first thought.Automobility has several factions that claim to define this system of automobility (3).   First is the socio-technical facet that defines automobility as the organization of modern concepts.   The political definition is the regulation and practices of movement.Others see it as the legitimization of the differences between man and machine, and man's dominance over the machine (3).   The one concept that is equivalent in all these definitions is the fact that automobility is a patterned system that creates a cohesive understanding of both autonomy and mobility (4).Upon a closer look, it becomes obvious that this system is really a regime based on truth, power, and subjectivity (8). To understand automobility in these terms, we must define the concept of mobility.   Universally it would be considered an automobile, but other modes of travel are available, such as bicycling, public transportation and taxis, and travelling by telephone and internet.These are all modes of travel, and are all self-regulated by the individual, thus giving them all the title of automobility, however, the car is the most often associated with this concept (6).Truth in this regime of automobility is the concept of driving the car (6).   Power is the brute for of the vehicle, such as trucks and sport utility vehicles which over power most other vehicle and modes of transportation (7). Another part of power are the governmental regulations and rules that accompany the driving of a vehicle, which has created a large amount of govern mental bodies to help keep the driver moving within the laws of automobility (7).While automobility is based in an individual’s freedom of movement, it also causes more regulations to be put on the individual and thus is the power of automobility (7). Subjectivity is the last part of the regime.The subjectivity can be a form of hierarchy or of self-monitoring.   Either way, the ideas of one group or class get put on others.   For example, cyclists are not considered when planning for roads and highways, thus those that drive cars, trucks, and such are putting their views and using their power.Social stigmatisms can also be place in this part, because the subjectivity of a person who views people taking public transportation as failures can lead to incorrect assumptions (8). In any case the three facets of the regime create the concept of automobility (8).There are four problems associated with automobility that affect the world at large.   First problem is the congestio n created with the number of cars and vehicles on the road and the planning of those roads being inadequate (9).The second is the ecological and environmental effects of automobility (9).   Third is the geopolitical factor that causes governments to spend money to find, store, and process the oil needed for the vehicles as well as the possibility of war to ensure the oil supply keeps coming.   The last problem is the deaths and accidents that occur, but are considered a normal part of automobility (10).The fact of the matter is that the concept automobility is virtually and completely impossible, since the concept itself requires external forces to allow it to be used.   For example, roads need to be created and maintained, licensure and driver education to ensure the regulations of the roads are followed, and even walking require sidewalks (11-12).Therefore, automobility is neither mobile nor individually self-regulated.   In fact the governmental bodies created by this con cept are now changing the rules and trying to alleviate or eliminate some of the problems associated with automobility (13 – 14).   In any case, changes are coming and the fragility of automobility has become more obvious, and yet the concept continues.

How to Type Accents in Italian on a Keyboard

Suppose youre writing to an Italian friend, and you want to say something like  Di dov’à ¨ la tua famiglia?  (Where is your family from?), but you don’t know how to type the accent over the â€Å"e.† Many  words in Italian  need accent marks, and while you could just ignore all those symbols, it’s actually quite easy to type them on a computer keyboard. You only need to make a few simple adjustments to your computers keyboard program—whether you have a Mac or a PC—and youll be able to insert accented Italian  characters (à ¨, à ©, à ², à  , à ¹) for  any electronic message. If You Have a Mac If you an  Apple Macintosh computer, the steps for creating accent marks in Italian are quite simple. Method 1: To place an accent over: à   option tilde (~) / then press the ‘a’ keyà ¨ option tilde (~) / then press the ‘e’ keyà © option ‘e’ key / then press the ‘e’ key againà ² option tilde (~) / then press the ‘o’ keyà ¹ option tilde (~) / then press the ‘u’ key Method 2: Click on the Apple icon on the top left of the screen.Click System Preferences.Choose Keyboard.Choose Input Sources.Click the add button on the bottom left of the screen.Choose Italian.Click Add.In the top right-hand corner of your desktop, click on the symbol of the American flag.Choose the Italian flag. Your keyboard is now in Italian, but that means you have a whole new set of keys to learn. Semicolon key (;) à ²Apostrophe key (‘) à  Left bracket key ([) à ¨Shift left bracket key ([) à ©Backslash key (\) à ¹ You can also choose Show Keyboard Viewer from the flag icon drop-down to see all of the keys. If you have a PC Using Windows 10, you can actually turn your keyboard into a device that will type Italian letters, accent marks and all. Method 1: From the desktop: Choose Control PanelsGo to the Clock, Language, Region option.Select (click on) Add a LanguageA screen with dozens of language options will appear. Choose Italian. Method 2: With the NumLock key on, hold down the ALT key and strike the three- or four-digit code sequence on the keypad for the desired characters. For example, to type à  , the code would be â€Å"ALT 0224.† There will be different codes for capitalized and lowercase letters.Release the ALT key and the accented letter will appear. Consult the Italian Language Character Chart for the correct numbers. Tips and Hints An upper-pointing accent, as in the character à ¡, is called laccento acuto, while a downward-pointing accent, as in the character à  , is called laccento grave. You may also see Italians using an apostrophe after the letter e instead of typing the accent above it. While this isn’t technically correct, it’s widely accepted, such as in the sentence: Lui e’ un uomo simpatico, which means, He’s a nice guy. If you want to type without having to use codes or shortcuts, use a website, such as this one from  Italian.typeit.org, a very handy free site that provides typing symbols in a variety of languages, including Italian. You simply click on the letters you want and then copy and paste what youve written onto a word-processing document or email.