The chemistry of lyophilized blood products - Bioconjugate Chemistry

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The chemistry of lyophilized blood products Joseph Fernandez-Moure, Nuzhat Maisha, Erin B. Lavik, and Jeremy Cannon Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00271 • Publication Date (Web): 23 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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Bioconjugate Chemistry

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The chemistry of lyophilized blood products

Joseph Fernandez-Moure, MD1,†, Nuzhat Maisha2,† , Erin B. Lavik, ScD2, Jeremy W. Cannon, MD, SM1,3,§

1

Division of Trauma, Surgical Critical Care & Emergency Surgery, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104 2

Department of Chemical, Biochemical & Environmental Engineering, University of Maryland, Baltimore County, Baltimore, MD, 21250 3

Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814

† Authors contributed equally to the writing of this manuscript § Corresponding author

Abstract With the development of new biologics and bioconjugates, storage and preservation have become more critical than ever before. Lyophilization is a method of cell and protein preservation by removing a solvent such as water from a substance followed by freezing. This technique has been used in the past and still holds promise for overcoming logistic challenges in safety net hospitals with limited blood banking resources, austere environments such as combat, and in mass casualty situations where existing resources may be outstripped. This method allows for long-term storage and transport but require the bioconjugation of preservatives to prevent cell destabilization. Trehalose is utilized as a bioconjugate in platelet and red blood cell preservation to maintain protein thermodynamics and stabilizing protein formulations in liquid and freeze-dried states.


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Biomimetic approaches have been explored as alternatives to cryo- and lyopreservation of blood components. Intravascular hemostats such as PLGA nanoparticles functionalized with PEG motifs, topical hemostats utilizing fibrinogen or chitosan, and liposomal encapsulated hemoglobin with surface modifications are effectively stored long term through bioconjugation. In thinking about the best methods for storage and transport, we are focusing this review on blood products that have the longest track record of preservation and looking at how these methods can be applied to synthetic systems.

Introduction Exsanguination can quickly occur in the setting of trauma. In the civilian and combat environment, it is the leading cause of preventable death. The central foci of hemorrhage management are bleeding control, restoration of oxygen carrying capacity, and clotting capability1, 2. Concomitant to pre-hospital hemorrhage control, is the implementation of damage control resuscitation (DCR). DCR is characterized by the minimization of crystalloid fluids, hemostatic (balanced) resuscitation, and selective permissive hypotension. Central to this paradigm is the use of hemostatic resuscitation2-4. In the pre-hospital environment, resuscitation with conventional blood products proves logistically difficult given the storage requirements and short shelf life5, 6. Further, storage lesion and functional decay of stored blood products can lead to negative effects on the microcirculation. Five-day shelf lives and stringent storage conditions make traditional blood products impractical in austere military environments that are commonly remote and in hot climates 7. The increasing need for blood products in both military and civilian settings has lead



to techniques for blood preservation to adapt to these environments. In particular, cryo- and lyopreservation techniques have enabled blood product transport and use in these extreme conditions 3. With the advent of novel bioconjugates, functional biomaterials, and new nanotechnologies with the potential for translation and clinical application, the question of preservation and storage is looming over the field. For many formulations, the lack of effective long-term storage could limit or stop clinical translation. In light of this, we have reviewed the preservation of complex systems, most notably blood products, to gain insights into the state of the art for preserving the function of bioconjugates. A brief history of pre-hospital blood transfusion The issues of remote damage control resuscitation are not new and solutions to overcome the logistical issues of blood product maintenance have been studied for decades. Freeze-dried (lyophilized) plasma (FDP) has been used since World War II (WWII). In addition to FDP, investigators have developed methods for red blood cell (RBC) and platelet (Plt) lyophilization and long-term storage.5. Although the risks of hepatitis and other disease transmission curtailed its use in the US, other countries such as France, Germany and South Africa have continued to both produce and use FDP on the battlefield. Today, component blood product administration is the standard of care, but the US has recently equipped special forces units with FDP from our French allies. Other products preserved by lyophilization are investigated but not currently available in the US. While we continue to separate and cold store our blood products, many would agree that the current methods for transfusion and preservation of this limited resource are in need of modernization.


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Principles of Lyophilization Lyophilization, also known as freeze-drying, is a method used for achieving stable formulations by removing solvent such as water, without disturbing the structure or function of the compound. The method is preferable in comparison to storing therapeutics in aqueous solution at room temperature to avoid degradation pathways such as hydrolysis, cross-linking, oxidation, aggregation and disulfide rearrangements. 8 Lyophilization begins with super-cooling for faster freezing of the aqueous solution. Drying then occurs in two steps. In the primary drying phase, sublimation takes place, leading to conversion of ice to water vapor, the driving force being pressure difference in the system. In the secondary drying phase, the unfrozen water is removed by evaporation as water diffuses from higher to lower concentration. 9 In Figure 1, an overview of the lyophilization process is presented. Ideally, the lyophilized product should be

Figure 1: A sketch of the lyophilization process occurring in three steps. Lyophilization begins with freezing of products, and after the sample is super-cooled, removal of water happens due to sublimation occurring during primary drying and desorption of water during secondary drying. This leads to formation of porous product cake.



able to retain porosity, avoid structural collapse, and have a high surface area to volume ratio for making rapid dissolution possible. Formulation composition plays a significant role in maintaining the structural integrity, as it determines the collapse temperature depending on individual glass transition temperature of the solute and excipients based on their mass fractions. 10

Cryoprotectants and lyoprotectants are used to prevent deactivation of proteins present during

freezing and loss of biological activity during drying. Two proposed mechanistic pathways include replacement of water by sugar during the drying step due to hydrogen bond formation between protein and sugar, or by vitrification during freezing. Table 1 lists common lyoprotectants and cryoprotectants used to stabilize formulations, along with their role in stabilization. Classification

Examples

Sugars

Lactose, maltose, trehalose, sucrose

Amino acids

Glycine, histidine, arginine

Polyols

Glycerol, sorbitol, mannitol

Polymers

Polyethylene glycol, dextran

Role of lyoprotectants and cryoprotectants I. II. III. IV. V.

Minimizing changes in conformation Formation of glassy state to immobilize proteins and solvent to prevent degradation Increase in chemical potential of proteins and additives due to cryoprotectants lead to unfavorable thermodynamic state. Leading to preferential exclusion of solutes from protein surface Strong correlation between increase in preferential exclusion and effectiveness of cryoprotectants

Table 1. List of commonly used lyoprotectants and cryoprotectants in lyophilization 11 These sugars, amino acids and polyols help by providing thermodynamic stability, higher glass transition temperatures, or by stabilizing protein formulations in liquid and freeze-dried states due to fast formation of an amorphous glass phase. 12 Some of these sugars are effective cryoprotectants due to a cell’s capacity to uptake them, leading to higher cryo-survivability. 13 1416

Thus, cryoprotectants and lyoprotectants work towards maintaining effective lyophilization

without affecting the therapeutic biological activity. Plasma Preservation


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In trauma patients with massive exsanguination, fresh frozen plasma (FFP) provides a bolus of coagulation factors to replace shed factors and to augment hemostasis at the site of injury. 17-20 Lyophilized plasma is of interest as it is possible to store the formulation at room temperature, and hence makes it a suitable choice in resource-limited situations. Dried plasma products are not yet approved by Food and Drug Administration for use in the United States, however, the Department of Defense (DoD) and Biomedical Advanced Research and Development Authority are working on developing dried plasma along with clinical trials to establish their efficacy and similarity to fresh frozen plasma. 21, 22 Lyophilized plasma manufactured by the French army is compatible with any blood type, as it is from pooled blood of more than ten donors, followed by selective neutralization of anti-A and anti-B antibodies. 23 A photochemical inactivation step has been made part of the process to further reduce risk of disease transmission. 24 Table 2 summarizes several studies performed on lyophilized plasma, characterizing the coagulation factors. Based on the studies, long-term storage at



Table 2. Summary of studies on lyophilized plasma25 26 27 28 29 30 31 32 33 elevated temperatures can reduce the activity of coagulation factors and inhibitors compared to fresh frozen plasma although the efficacy of lyophilized plasma is similar to fresh frozen plasma. 25, 29, 32

An emerging concept is to substitute the drying steps described above with spray drying, which unlike lyophilization, requires higher temperature exposure for evaporating water from product droplets. While coagulation factors were slightly lower compared to fresh frozen plasma for spray dried plasma resuspended in deionized water, resuspension in 1.5% glycine showed


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similar profile for coagulation factor compared with fresh frozen plasma. Optimum reconstitution methods and potential immunogenicity need to be evaluated for future studies. 34 Red Blood Cell Preservation Lyophilized red blood cells (i.e. erythrocytes) and red cell substitutes eliminate cold storage requirements and offer extended shelf life. Key factors to be considered during lyophilization of erythrocytes include determining methemoglobin level, preservation of metabolic pathways and oxygen carrying capacity, and maintaining cell membrane flexibility upon re-constitution. Numerous studies have been performed to assess these essential aspects of erythrocyte lyophilization as summarized in Table 3.



Table 3. Summary of studies done on lyophilized erythrocytes 35 36 37 38 39 40 41 Optimized freezing temperature and freeze-drying temperature can lead to formulations with unaltered metabolic pathways and prevent hemoglobin loss. 36-38 Addition of cryopreservatives such as trehalose has been used and compared to formulations without any cryopreservatives. With such additives, lyophilized red cells have lower methemoglobin levels, very slightly altered quantity of metabolic enzymes, and preserved secondary structure of hemoglobin. 39 During freezing, irreversible structural change can happen due to displacement of water adjacent to cells. Preservative disaccharides such as trehalose can replace the displaced water and form hydrogen bond with cells, preventing protein denaturation and ice crystal formation. Moreover, while freezing, due to changes in membrane, cells can uptake trehalose. Zhang et al. found that for mammalian cells, DNA content remains least affected for lyophilized formulations with trehalose however recommended storage temperature was 4℃.

42

Among methods for increasing trehalose uptake, electroporation has been used,

however, hemolysis rate is even higher compared to lyophilized erythrocytes with trehalose without electroporation. 43 Synthetic biopolymer, PP50, was also used to bind to phospholipid cell membrane, bending hydrocarbon chains, and increasing trehalose uptake. This method showed decrease in hemoglobin oxidation with increase in trehalose uptake, i.e. PP50 attachment


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to phospholipid membrane (figure 2) .44

Figure 2. Association with PP50, a pH responsive polymer can lead to higher trehalose uptake, until reaching concentration above which effect on cellular uptake of PP-50 by red blood cells is diminished (a), however increasing polymer concentration can lead to higher bi-layer thinning leading to cell membrane collapse (b). At 200 ߤg/mL PP-50 concnetration and pH 7.05, increase in trehalose uptake lead to increase in membrane integrity (c) and decrease in hemoglobin oxidation rate (d). 44 All these suggest that optimized formulations with lyoprotectant can produce stable lyophilized erythrocytes with least affected hemoglobin content and metabolite content. Compared to cryopreserved deglycerolized RBC this allows for the stable storage of RBC at room temperature in a powdered form. Renewed interest in whole blood transfusion for acute hemorrhage resuscitation has emerged in recent years.2, 45, 46 Fresh whole blood is used by the US military during combat



operations. Although this practice is currently considered “off label” by the Food and Drug Administration (FDA), it appears to have a very favorable mortality benefit with an extremely low risk of disease transmission or transfusion reactions45, 47. In the civilian setting whole blood is stored in citrate phosphate dextran anticoagulant to prevent RBC aggregation. The use of citrate phosphate dextran, though, has been shown to impair tissue oxygenation48. To prevent settling of blood cells, preservation with Ficoll 70 kDa has been shown to prevent RBC aggregation and maintain leukocyte viability49. This method is more relevant for the preservation of whole blood for ultimate component separation rather than whole blood transfusion. While lyophilized whole blood has been investigated as a method to preserve blood for HLA molecular typing, a limiting factor for effective preservation is the RBC stability. No studies to date have used lyophilized whole blood for acute resuscitation of patients in hemorrhagic shock50. This presents an opportunity for innovation and development. Platelet preservation Platelets are essential to hemostasis through the formation of an organized clot. This occurs by the sequential binding to exposed receptors on the damaged endothelium and the release of the many coagulation factors they store. In order to achieve local hemostasis the platelet must remain intact and thus researchers have sought ways to preserve the procoagulant properties of the platelet following lyophilization or cryopreservation. Lyophilized platelets were first developed to evaluate the effectiveness of von Willenbrand factor in clinical assays 51. Platelets are exquisitely sensitive to chilling. When stored below 20°C they change their shape with filamentous actin reorganization, increase intracellular calcium and can undergo secretion of alpha granules mimicking physiologic activation52-54. Preservatives such as Me2SO, hydroxyethyl starch, glycerol-glucose, and Me2SO


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with second messenger effectors have been studied55, 56. Me2SO emerged as superior in preservation but had poor in-vivo recovery and high clearance by the reticuloendothelial system 57, 58

. Trehalose, as mentioned above, is capable of increasing cell tolerance to dehydration and is

used as a cryo and lyoprotectant 15, 59. The mechanism by which trehalose allows for platelet preservation is still unknown. The “water replacement hypothesis”, as mentioned above, states that trehalose replaces water in forming hydrogen bonds with proteins and membrane lipids during dehydration 60. An alternative hypothesis suggests that trehalose forms an amorphous glassy matrix instead of the formation of ice crystal during the preservation. When compared to another small carbohydrate such as sucrose, trehalose has a higher glass transition and its dehydrate crystalline structure may explain its improved lyophilized stability60, 61. Preservation of biologic function is paramount in the investigation of platelet preservation. Platelets lyophilized with trehalose as a protectant have not only been shown to survive with a survival rate of 85% but maintain thrombogenic capabilities in response to thrombin, collagen, and ristocetin (Figure 3) 15. These properties have played out in vivo



Figure 3. Platelets lyophilized with trehalose as a protectant have not only been shown to survive with a survival rate of 85% but maintain thrombogenic capabilities in response to thrombin, collagen, and ristocetin 15 where species specific lyophilized platelets were shown to be capable of clot formation and decrease of blood loss 62. These functional properties make lyophilized platelets an attractive alternative to the current standard of care. Cryoprecipitate and Fibrinogen Preservation Cryoprecipitate, originally developed as a therapy for Hemophilia A, has been in use for almost 50 years63. It is prepared from FFP through a process of thawing followed by centrifugation to collect a “precipitate” of insoluable factors such as fibrinogen, Factor VIII, von Willenbrand factor, and Factor XIII63.Its most common clinical application is the acute replacement of depleted fibrinogen levels in bleeding patients. Before the advent of factor concentrates for treating congenital clotting factor deficiencies, lyophilized cryoprecipitate was used in the treatment of patients with Heamophilia A and without inhibitor to Factor VIII C 64, 65. Lyophilized cryoprecipitate was shown to restore effective Factor VIII clotting activity. More recently, lyophilized fibrinogen concentrate has been investigated and used predominantly in cardiac surgery66. The use of lyophilized fibrinogen concentrate is considered safe and has been investigated in multiple randomized trials66-70. Unfortunately the use of lyophilized fibrinogen concentrate in these small studies did not result in a significant difference when compared to conventional treatment of cardiopulmonary bypass induced coagulopathy66. Biomimetic Biosynthetic Alternatives to Resuscitation in Austere Environments Hemostatic Nanoparticles


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Nanomaterials have emerged as promising alternatives to current blood products or even their lyophilized counterparts. Several groups utilize the specific component interactions of the coagulation system for developing targeted hemostats. Lyophilized polymeric nanoparticles conjugated with peptide containing arginine-glycine-aspartic acid (RGD) mimic fibrinogen activity, leading to reduced bleeding times in rodents by up to 50% following femoral artery injury. Moreover, poly-lactic acid core instead of previously used poly-lactic-co-glycolic acid makes it suitable for storing at room temperature without decreasing hemostatic efficacy. 71, 72 Platelet like nanoparticles of discoidal shape decorated with ligands have been reported to reduce bleeding time by 65%. 73 Liposome nanovesicles with PEG linker joining peptides that can bind to activated platelet integrins GPIIb-IIIa and P-Selectin have been assessed for clot formation. However, these require storage at 4℃ in aqueous solution, and the presence of complex ligands complicate long-term storage limiting clinical translation. 73, 74 Another emerging synthetic material is the POLYSTAT, that acts like FXIIIa and crosslinks with fibrin for increasing clot stiffness. The synthetic polymer is lyophilized, hence can be stored long-term and administered by first responders. Once patient is stabilized, though, the clot must be surgically removed. Compared to human FXIIIa, POLYSTAT can reduce hemorrhage volume. 75 There are several topical hemostatic materials utilizing fibrinogen, fibrin, chitin, chitosan, or mineral zeolites for concentrating clotting factors and continuing the healing process. 76 Mentioned studies mainly focused in synthetic blood products directed towards intravenous administration. As these are administered intravenously, size of these are of higher importance, as aggregation or degradation of biomolecules during lyophilization process can provoke adverse biological reactions in immune system as well as reduce efficacy of the treatments. Artificial Oxygen Carriers



The primary function of hemoglobin (Hb) is to transport and deliver oxygen to the tissues. Artificial oxygen carriers have been investigated as alternatives to RBCs, given the aforementioned difficulties in austere environments for blood maintenance. Several carriers have been developed and can be grouped broadly into -based materials, synthetic Fe2+ porphyrinbased materials, and hemoglobin (Hb) based materials 77-80. Hemoglobin is comprised of four polypeptide chains with Fe2+ protoporphyrin IX as a prosthetic group. Logically, the isolation and encapsulation of cell-free Hb was sought as a potential solution. Unfortunately, cell-free quaternary Hb was found to dimerize in the absence of a cell and be rendered inactive. In order to overcome this, bioconjugative methods were sought. Polyethylene glycol conjugation, polysaccharide conjugation, and glutaraldehyde polymerized Hb have been studied but fraught with in vivo complications such as renal toxicity or the binding of nitric oxide or carbon monoxide, inducing vasoconstriction and paradoxical lowering of oxygen delivery to the tissues80-82. To avoid this, these free Hbs were encapsulated to preserve the oxygen carrying activity and prevent undo monoxide binding. Bioinspired methods such as polydopamine (PDA) coated hemoglobin microcapsules have demonstrated oxygen carrying and antioxidant properties83, 84. Encapsulation of Hb in PDA was found to have oxygen radical scavenging properties and minimal influence on blood constituents and cell viability83. Liposomal Hb carriers have also emerged as promising encapsulation agents but because of their inherently unstable nature required stabilization 85. Polymerization of phospholipids with 1,2Dioctadecadienoyl-sn-glycero-3-phosphotidyl choline, for example, was capable of generating stable liposomes resistant to macrophage attack up to 30 days in vivo 81, 86. Alternatives to liposomes have also been investigated because of their increased stability.


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PEG conjugation to a lipid backbone has been synthesized and proposed as a more stable alternative to liposomal Hb carriers. PEG conjugation offers the ability to load large amounts of Hb into vesicles because of the volume exclusion effect of the outer surface PEG80. Complement activation is an unfortunate complication of the injection of liposomal Hb seen in previous studies. In order to overcome this issue a minimal amount of a negatively charge phospholipid is required. PEG-modification and a negatively charged lipid DHSG have been able to maintain compatibility with blood and not cause thrombocytopenia or pulmonary hypertension, commonly seen reaction to other encapsulated Hbs 80, 87. PEG modification of phospholipids has enabled a prolonged circulation time through decreased reticulo-endothelial system uptake. These approaches have ushered this innovative solution closer to clinical reality. Stabilizing Polymeric Nanomaterials through Lyophilization Freeze-drying or lyophilization of polymeric nanomaterials and bioconjugates can result in concentrated formulation, and hence would be beneficial in determining dosages. Moreover, it has higher shelf life and can be stored at elevated temperatures. However, it is possible to lose stability of suspensions after freeze drying due to van der Waal’s interaction between particles, depending on its surface charge. 88 Surface modification by adding ionic surfactants can prevent aggregation, undesired protein adsorption on surface or macrophage clearance that can cause lower biodistribution. 89, 90 Common destabilization phenomena that can occur in nanoparticles include the displacement of steric stabilizing surfactant layer from polymeric nanoparticles, or flocculation due to bridging in a dilute dispersion if a high molecular weight polymer is added. 91 Moreover, type of polymer, pH, entrapped drugs and storage temperature can influence chemical stability of lyophilized nanoparticles. 91 Lyophilization of polymeric nanoparticles is not much different from general lyophilization process, where an amorphous glassy state goes through



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sublimation to remove frozen water. Cryo and lyoprotectants are added to either bulk up concentration for instances when concentration is very low, or for adjusting pH, protecting against drying or freezing stresses, or to increase collapse temperature of formulation. 91 There is much debate whether a fast freezing or slow freezing condition is better for nanoparticles suspensions. Increase in cryoprotectants molecular weight also can increase nanoparticles’ redispersion. As most of the examples mentioned in this document are in lab-scale, scaled up nanoparticle lyophilization systems are still lacking. 92 Table 4 presents a list of cryo and lyoprotectants used for freeze drying polymeric nanomaterials and bioconjugates. 90, 93-99

Protectant

Polyacids (polyacrylic acid, citric acid)

High molecular weight PEG

Mechanism/purpose

• Coacervate precipitation due to hydrogen bonding between oxygen in surface peg group and hydroxyl ion in carboxylic part of polyacid. • This method is of interest due to possibility of using hetero bifunctional PEG in for binding with biomolecules of interest. • PEG between 0.4-20 kDa used as cryoprotectant at different freezing rates. • Higher particle size compared to initial size represents higher order of aggregation.

Surface stabilization with quaternary ammonium groups

• As a second cryoprotectant lactose, microcrystalline cellulose or calcium phosphate was used. • Four model polymeric nanoparticle were investigated. • The surface stabilization through ammonium group caused low contact angles.

Polyvinyl alcohol (pva)

• Freeze drying of polycaprolactone nano-capsules in presence of pva • Comparing observations against sugars (glucose, mannitol, trehalose) used for cryoprotection

Pluronics

• Role of poloxamer 188 was investigated • Surfactants such as these are supposed to be adsorbed in surface of polymer nanoparticles

Outcomes • In presence of high amount of sucrose, the nanoparticles resuspended, while for nanoparticles without sucrose, 15 min sonication was required. [77]

• Aggregation was highest in low molecular weight peg in comparison to suspension with high molecular weight peg. Freezing rate did not affect the rate of aggregation. [78] • Higher wettability of polymer surface and high minimum film formation were attributed for better redispersion of polymeric nanoparticles released from granular, pelleted or tablet formulations. [79]

• Using 5% w/v pva resulted in almost complete resuspension of nanoparticles similar for freezing in liquid nitrogen, in pre-chilled freezer, and freeze dryer shelf. [80] • 5% poloxamer solution used lead to ratio of final and initial radiuses close to 1.1, while combining glucose resulted in ratio close to 1. [74] • Lower flocculation and higher efficacy for delivering biomolecules [81]

Trehalose

Lactose and sucrose

Dextran

• Using trehalose to modify solvation layer around biomolecules due to larger hydrated volume. • Designed polymers were formed from diazide-trehalose comonomer and dialkyne comonomer • Flocculation levels were studied in media containing serum proteins • Formation of glassy layer around particles to prevent aggregation. • Studying change in physical properties of lyophilized cake with variation in amount of sugar cryoprotectants for cationic nanoparticles

• Formation of stabilizing layer on particles, resulting in an interface between water and the latex particle

• In absence of cryoprotectants, higher freezing temperature lead to higher flocculation. Redipersion at 5% w/v of sugar used resulted in size closest to initial particle size, and as amount of sugar was increased, particle size went up as well. [82] • As amount of dextran increases, layer thickness on particles increases, and size when re-dispersed after freeze drying becomes closer to initial size. • As molecular weight of dextran increases, lesser amount of dextran (w/w) is required for better resuspension of latex particles. [83]

Abbreviations: polyethylene glycol (PEG)

Table 4: List of Lyoprotectants and cryoprotectants used for lyophilization of bioconjugates [74] 93, 94 95-98 99


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Sugars like fructose, glucose, trehalose, lactose, sorbitol etc. form amorphous glassy layer preventing sticking between particles and allowing greater dispersion (Figure 4). 98

A

B

C

Figure 4. (a) For oral delivery of polymeric nanoparticle drugs, lactose as excipient can redisperse the nanoparticles completely in granular and tablet forms100 (b) Use of Polyacrylic acid for coacervate precipitation resulted in nanoparticle diameters closer to initial diameter for freeze dried formulations, and got even closer to initial diameter with longer dispersion time101. (c) Styrene nanoparticles with high molecular weight dextran (T5000) shows least particle aggregation compared to coatings of lower molecular weight dextran (T6 and T40)102 For Insulin loaded in poly-lactic-co glycolic acid nanoparticles, using sugars as cryoprotectants resulted in particle size similar to initial size after lyophilization. 103 Use of surfactants such as Polyacids (PVA, Pluronics, Tween) can also reduce aggregation by stabilizing the suspensions as these are adsorbed on the surface of nanoparticles. 93, 95, 96, 103 However, for surfactants such as poloxamer that have higher solubility in lower temperatures of water, during freezing, destabilization can occur. 90 The level of aggregation leading to an increase in nanoparticle size depends on the cryo or lyoprotectant used and in many cases, requires sonication to retain the initial size. Sonication uses ultrasonic sound waves to disrupt



Table 5. Oligosaccharides and pluronics used as protectants during lyophilization 90 agglomerates but leads to increase particle collisions and paradoxically can lead to agglomeration. Combining surfactants with cryo and lyoprotectants can enhance the freezedrying process resulting in better redispersion, and thus preservation of particle radius pre and post-lyophilization (Table 5). 90, 96 Depending on materials properties, pressure and temperature gradients and shock waves can result in aggregation as well. Specifically, for bioconjugates that have proteins conjugated, the organic molecule can go through irreversible degradation leading to adverse biological reactions. 104 Taurozzi et al. have outlined a number of considerations for standardized reporting of sonicating applications for engineered nanoparticles. 104 The aim in lyophilizing polymeric formulations with biomolecules conjugated is to concentrate the formulation for easier dosages, better shelf lives and higher storage temperatures. Combining cryoprotectants and surfactants can help in achieving final and initial radius of


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particle ratios much closer to 1. 90, 96 An optimized lyophilization process with rightly chosen protectants and surfactants can help in overcoming some of the difficulties associated with aggregation post-lyophilization, help preserve bioactivity and physical characteristics for the formulation as well as avoid adverse reactions in the body. Conclusions The transport and maintenance of blood products in the military theater has been limited by the portability of blood components and their ability to tolerate warm temperatures. As early as WWII, the use of lyophilization emerged as a suitable method for blood product preservation and almost 80 years later, this method of storage remains an appealing approach due to its logistical advantages. Use of sugars, polymeric excipients and surfactants alone as well as in combination have made long-term cryo- and lyopreservation a reality. Likewise, continued progress in nanomaterial synthesis carries a significant potential for alternatives to conventional blood product preservation. While many of these advancements have yet to make it into the clinical armamentarium the possibilities are vast and greatly anticipated.

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Chang, R., Eastridge, B. J., and Holcomb, J. B. (2017) Remote Damage Control Resuscitation in Austere Environments. Wilderness Environ Med 28, S124-S134. Cannon, J. W. (2018) Hemorrhagic Shock. N Engl J Med 378, 370-379. Daniel, Y., Habas, S., Malan, L., Escarment, J., David, J. S., and Peyrefitte, S. (2016) Tactical damage control resuscitation in austere military environments. J R Army Med Corps 162, 419-427. Cannon, J. W., Khan, M. A., Raja, A. S., Cohen, M. J., Como, J. J., Cotton, B. A., Dubose, J. J., Fox, E. E., Inaba, K., Rodriguez, C. J., et al. (2017) Damage control resuscitation in patients with severe traumatic hemorrhage: A practice management guideline from the Eastern Association for the Surgery of Trauma. J Trauma Acute Care Surg 82, 605-617.



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The chemistry of lyophilized blood products - Bioconjugate Chemistry

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