Development of Hemosomal Drug Delivery System

Figure 2:  Hemoglobin encapsulated within liposomes.

Hemoglobin vesicles or liposome–encapsulated hemoglobin (LEH) mimics membrane enclosed cellular structure of red blood cells. Compared to free modified hemoglobin preparations, LEH is characterized by spatial isolation of hemoglobin by an oxygen permeable lipid layer that eliminates the free modified toxicity. With that, co–encapsulation of reductants antioxidative enzymes and oxygen–affinity modifiers inside liposome’s is possible to enhance resuscitative capacity of LEH [6]. The lipid bilayer of encapsulated hemoglobin prevents renal clearance, extra vacation and direct contact of hemoglobin with vascular smooth muscle, thus prevent vascular constriction, increasing the in vivo half–life of LEH [7]. Also this membrane reduces interactions between hemoglobin and nitric oxide, adverse effects such as hypertension and histological damage in myocardial lesions, are not induced, as are found for a cellular–type HBOCs (hemoglobin–based oxygen carriers) [8].

The Success and progress in the generation of encapsulated hemoglobin can be improved by following considerations:

1. Increasing storage stability and then infusion.
2. Effects of lipid decreasing on the reticuloendothelial systems.
3. Avoiding peroxidation of lipid.
4. Solving the problem of met hemoglobin formation.
5. Inclusion of most of functioning red blood cell enzymes [9].

In this work we report synthesis of a simple phospholipid to enhance hemoglobin encapsulation inside liposome’s.

A part of optimization of LEH formulation in this article we present synthesis of novel lipid and LEH formulation development as an oxygen carrier.

The overall goal of our work is the development of safe, efficacious and economically viable oxygen carrying red blood cell substitute composed of hemoglobin solution encapsulated in a liposome.

Hemosomes or LEH have many important advantages

Compared to un encapsulated hemoglobin LEH has many advantages which include the following:

1. Minimised nephrotoxicity.
2. Metabolism by RES (reticuloendothelial system) of the liver and spleen same as red cells [10].
3. Potential to Co–encapsulate Allosteric Modifiers and Antioxidants with Hemoglobin. During manufacture of LEH Allosteric modifiers can be co–encapsulated with the hemoglobin in order to control the oxygen affinity (P50) [11]. Hemoglobin protectants can also be encapsulated in the liposome in order to retain the hemoglobin in the oxy–hemoglobin state [12].
4. Decreased Vasoactivity, because physical properties of LEH have closer to red cells, it produces less of a hypertensive response than that observed with cell-free hemoglobin [13]. Recent studies demonstrate that the vasoconstrictor activity of LEH is 60 times less than that of un encapsulated Hb [14].
5. Diffusive Properties Closer to Red Cells. The rate of oxygen release from LEH is slower than from cell-free hemoglobin i.e. closer to the rate of release from intact red cells [15]. Rapid oxygen release from un encapsulated Hb may cause hypertension secondary to auto regulation at the level of the arterioles [16].
6. Decreased Likelihood of Neurotoxicity, because of the protective lipid encapsulation of the hemoglobin with LEH [17].

The hemosomes or LEH formulation produced by following characteristics

1. It is a homogeneous LEH formulation that is approximately 0.25 microns in diameter, unlike the originally described LEH formulation which contained large particles of > 1 micron (~ 30% of the population).
2. Due to the addition of albumin to the new formulation and the use of a PEG (poly ethylene glycol) coating now the LEH is a volume expander. Recent research demonstrates that intravascular volume expansion is a very important additional aspect of a resuscitative fluid (i.e. no oxygen transport is possible without adequate intravascular volume) [18].
3. By addition of PEG to the LEH formulation has increased the circulation half-life of LEH from 18 hours up to 65 hours [19].
4. Prior LEH and other liposome formulations have been reported to cause an acute thrombocytopenic [20]. Coating the surface of LEH with PEG as well as greatly reducing the negative lipid component from 10% to 2% of the formulation has also greatly reduced the thrombocytopenic response in small animals as studied.

Progress has been limited by three major obstacles

We have made considerable progress in all three following fronts.

1. Selecting an acceptable microencapsulation material;
2. Developing a microencapsulation process that yields the desired size range, but avoids denaturation of Hb; and,
3. Encapsulating sufficient Hb while maintaining an acceptable final viscosity [21].

Materials used for LEH preparation

Egg Phosphatidylcholine (EPC), Dimyristoyl Phosphatidylcholine (DMPC) and bovine Brain Phosphatidylserine (BPS; horse heart cytochrome C, the sodium salt of diphosphoglyceric acid and tetra-methylene phenylenediamine; cholesterol and Dicetyl Phosphate (DCP), Inc. Hemoglobin derived from outdated human blood at - 150 mg/ml Cs. All other chemicals were reagent grade and used without additional purification. Polycarbonate membranes were obtained from Nucleopore [22,23].

Techniques for Hemosomes or LEH preparation

Aseptic techniques are followed in the preparation of hemosomes; the widely used techniques are included such as,

1. Film hydration followed by sonication,
2. Membrane extrusion,
3. French press extrusion,
4. Micro fluidization or homogenization,
5. Detergent dialysis,
6. Reverse-phase evaporation, reverse- micelle: and other mechanisms.
7. Most recently approach is multiple emulsions [24].

Among above all mainly three methods are used for producing LEH:

1. Film hydration,
2. Reverse evaporation and
3. Double emulsion.

Film hydration

This method used to form multilamellar vesicles, which are then reduced in size by a Micro-fluidizer TM M1 10 (Micro fluidics. Newton, Ma). Most recently modified this process (prior to Micro fluidization) by rotary evaporation (operating under vacuum at room temperature) of as much water as possible from the formed LEH in Hb solution. This results in a somewhat dry Hb/LEH film that deposit on the walls of a round bottom flask. Hb solution is added to rehydrate and re suspend the LEH. Micro fluidization processing at temperatures of about 5°C follows. The LEH so formed are washed at least three times in isotonic phosphate buffered saline(PBS) and centrifuged at 20,000 X g for 20mins to remove all un encapsulated Hb solution and any remaining organic solvent (if any). The washed liposome’s are then re suspended in isotonic PBS containing 7.5 g% egg albumin [25,26].

Reverse-phase evaporation

In this technique, sonication is not used as it damages the hemoglobin. The approach used here in involves formation of an emulsion of aqueous concentrated Hb solution in an organic solvent mixture of diethyl ether and trichlorotrifluoroethane, which contains in dissolved form the formulated phospholipids/ lipids. Organic solvents are slowly evaporated at room temperature in a rotary evaporator operating under partial vacuum, as the organic solvents are removed. LEH spontaneously form in the excess lipid system. The evaporation procedure is continued until dryness to maximize removal of all organic solvent and water so that the Hb concentration within the LEH is as high as possible. This results in the deposition of an apparently dry Hb/LEH film on the walls of the round bottom flask. Concentrated Hb solution is then added under agitation to rehydrate and re suspend the LEH. Micro fluidization, washing and re suspension procedures used for LEH are the same as described above for film hydration [27,28].

Double emulsion

In this method the formation of a water-in-oil-in- water type multiple emulsion with removal of organic solvent and then size reduction in a Micro fluidizer to form LEH . First a primary emulsion is formed of Hb solution droplets dispersed in an organic solvent blend containing in dissolved form the formulated lipids/ phospholipids. Components and compositions are exactly the same as used above for reverse evaporation. The primary emulsion is then well-dispersed into precursor Hb solution to form the double emulsion. The remaining steps in the process to form LEH that involve organic solvent removal, micro fluidization and washing are similar to those reported above for the reverse evaporation method [29,30].

Method of preparation of LEH

The solvent (chloroform and methyl alcohol) is subjected to remove with a rotary evaporator from 75 pmol of a mixture of EPC, cholesterol and BPS 5: 4: 1 or of DMPC, cholesterol and DCP 5: 4: 1. Concentrated hemoglobin (3 ml) in buffer (100mM NaCl, 20mM HEPES, 1mM EDTA, pH 7.4) is added to the lipid, which is dispersed by overtaxing. The mixture is then placed at 20°C in an ultra-filtration cell (Amicon Model 12) and forced by argon pressure through polycarbonate membranes twice each through membranes with 6000 A, 4000 A and 2000 A pores. The resulting mixture of vesicles and free hemoglobin is separated at 4°C on a column of Sepharose CL-6B (Pharmacia). The purified vesicles may then be concentrated on an, & 100000 cut-off ultra filter (Nucleopore), or by centrifugation at 5000 rev/min for 20 min [31] (Figure 3).

Figure 3:  Manufacturing technique of Liposome-Encapsulated Hemoglobin (LEH) preparation.

  

    
Title    of patent
    
Inventors
    
Innovation
    
Patent no.
    
Reference
  
  

    
Lipid    encapsulated hemoglobin cells
    
Irving F. Miller et al. 
    
Synthetic    erythrocytes, Lipid encapsulated hemoglobin cells preferably cholesterol one    or more phospholipids are included
    
US4133874 
    
36
  
  

    
Hemoglobin    containing liposomes and process for preparing the same
    
Suzuki,    Kazuhiko et al.
    
Hemoglobin    containing liposomes and process for preparing the same by thin film process    comprising liposome forming lipid material in organic solvent
    
EP    0170247 B1
    
37
  
  

    
Processes    for reconstituting lyophilized erythrocytes and/or hemosomes
    
Goodrich,    Raymond Paul et al.
    
Processes    for the reconstitution of lyophilized red blood cells and/or hemosomes    comprising the use of solutions including monosaccharide hexoses and    pentoses, and biocomparible amphipathic...
    
EP0392813    A2
    
38
  
  

    
Liposomes    on which adsorption of proteins is inhibited
    
Terumo    Kabushiki Kaisha et al.
    
Liposomes    on which adsorption of proteins is inhibited which are agglutination-free
    
0    354 855 B1
    
39
  
  

    
Storage-stable    lipid vesicles and method of preparation
    
Murray Weiner et al. [44] 
    
Storage-stable    lipid vesicles and method of preparationcontaining inositol hexaphosphate
    
4397846
    
40
  
  

    
Liposome    encapsulated hemoglobin
    
Kazuhiko Suzuki et al. [45]
    
The    invention relates to hemoglobin-containing liposomes in which an aqueous    solution of hemoglobin is incorporated comprising liposome membrane mainly    composed of hydrogenated phospholipids
    
5049391
    
41
  
  

    
Scaled-up    production of liposome-encapsulated hemoglobin
    
Martha    C. Farmer et al.
    
A    method for production of liposome-encapsulated haemoglobin comprising    chloroform, HSPC, cholesterol, DMPG, alpha-tocopherol
    
US4776991
    
42
  
  

    
Hemoglobin-encapsulated    liposome 
    
Ogata,    et al.
    
preparing    a hemoglobin-containing solution comprising a concentrated stroma-free    hemoglobin solution
    
5674528
    
43
  

Table 1:  Patent Technologies in Hemosomes.

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Citation: Idris NF, Hundekar YR, Chilkawar RN, Nanjwade BK, Kamble MS and Srichana T. Development of Hemosomal Drug Delivery System. Austin J Anal Pharm Chem. 2014;1(3): 1011. ISSN:2381-8913

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