Nanocochleate: A Review

 

Suraj R. Wasankar*, Kshitij V. Makeshwar, Abhishek D. Deshmukh, Rahul M. Burghate

Vidyabharti College of Pharmacy, Camp Road, Amravati, Maharashtra 444602. India

 

 

ABSTRACT:

The nanocochleate drug delivery vehicle is based upon encapsulating drugs in a multilayered, lipid crystal matrix (a cochleate) to potentially deliver the drug safely and effectively. Nanocochleates are cylindrical (cigar-like) microstructures that consist of a series of lipid bilayers. Nanocochleate delivery vehicles are stable phospholipid-cation precipitates composed of simple, naturally occurring materials, generally phosphatidylserine and calcium. They have a unique multilayered structure consisting of a solid, lipid bilayer sheet rolled up in a spiral or in stacked sheets, with little or no internal aqueous space. This structure provides protection from degradation for associated “encochleated” molecules  negatively  charged  lipids  and  drugs  or  peptides acting  as  the  inter‐bi‐layer  bridges  instead  of multi‐cationic  metal  ions.  This  new  type  of  cochleate  opened  up  to  form  large  liposomes  when  treated  with cationic salt, suggesting that cationic organic molecules can be extracted from these cochleates in a way similar to multivalent metal ions from metal ion‐bridged cochleates. Cochleates can be produced in sub‐micron size using a method known as “hydrogel cochleation” or simply by increasing the ratio of multivalent cationic peptides  over negatively charged liposomes. When nanometer‐sized cochleates and liposomes containing the same fluorescent labeled lipid component were incubated with human fibroblasts cells under identical conditions.

 

KEY WORDS: Cochleate, Bilayers, Phosphatidylserine, Encochleated, Peptides.

 

INTRODUCTION:

Various approaches have  been  reported  for  oral delivery  of  tissue-impermeable  drugs. For  example i)  converting  a  drug  to  lipophilic  pro-drug,  ii) conjugating  a  drug  with  lipophilic  moieties,  and  iii) encapsulating  a  drug  into  particulate  systems  (1). Particulate  systems  may  offer  good  protection  of delicate  biological  agents  with  no  need  for  chemical modification  of  the  molecules  themselves.  However, absorption  of  particles  by  the  intestines  is  generally less than 1% (1). Because of the structural similarity of liposomes  (phospholipid  bilayer  vesicles)  to  cellular membranes,  the  material  had  once  been  regarded  as an ideal system for delivering therapeutic agents and attracted considerable research interest (2). However, utilization  of  liposomes  to  improve  oral  absorption of  hydrophilic  and  hydrophobic  drugs  remains unsuccessful  mainly  due  to  their  poor  mechanical stability,  low-drug  loading  capacity,  and  probably the  lack  of  mechanism  to  facilitate  cross  membrane diffusion  in  intestine..  To overcome this  stability problem,  several  alternative  lipid  bilayer  systems have  been  reported,  namely,  stealth™  liposomes (2),  polymerized  liposomes  (3),  polyethylene glycol  coated  liposomes  (4),  lipo-beads  (5),  and cochleates (6).

 

The nanocochleate drug delivery vehicle is based upon encapsulating drugs in a multilayered, lipid crystal matrix (a cochleate) to potentially deliver the drug safely and effectively. Nanocochleates are cylindrical (cigar-like) microstructures that consist of a series of lipid bilayers (Fig.1, 2). (7)

 

Fig.1: Freeze-fracture electron                                                     

 

Fig. 2: Nanocochleate Structure

 

Nanocochleate delivery vehicles are stable phospholipid-cation precipitates composed of simple, naturally occurring materials, generally phosphatidylserine and calcium. They have a unique multilayered structure consisting of a solid, lipid bilayer sheet rolled up in a spiral or in stacked sheets, with little or no internal aqueous space. This structure provides protection from degradation for associated “encochleated” molecules. Because the entire nanocochleate structure is a series of solid layers, components encapsulate within the interior of the nanocochleate structure remain intact, even though the outer layers of the nanocochleate may be exposed to harsh environmental conditions or enzymes. (8,9) Because nanocochleates contain both hydrophobic and hydrophilic surfaces, they are suitable to encapsulate both hydrophobic drugs like amphotericin B and clofazimine and amphipathic drugs like doxorubicin. (7)

 

Route of Administration(10)

Efficient oral delivery of drugs. An alternative route of administration can be used. The various route of administration are as given in table no 1.

 

Table no 1- Route of Administration

Parenteral

Ophthalmic 

Intrathecal

Intrauterine

Rectal

 

Subcutaneous

 

Intra-Articular

Intravaginal

Topical

Intramuscular

IntraArterial

Lymphatic 

Sublingual

 

Intravenous

 

Sub

Arachniod

Nasal

Mucosal or any other mucosal surfaces

Transdermal

Bronchial

Spinal

 

Dosage form(11)

·         For oral administration: Capsules, cachets, pills, tablets, lozenges, powders, granules, or as a solution or a suspension or an emulsion.

·         For topical or transdermal administration: Powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.

·         For parenteral administration: Sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use.

 

Advantages:

1)       They are more stable than liposomes because the lipids in nanocochleates are less susceptible to oxidation. They maintain structure even after lyophilization, whereas liposome structures are destroyed by lyophilization.

2)       They exhibit efficient incorporation of biological molecules, particularly with hydrophobic moieties into the lipid bilayer of the cochleate structure.

3)       They have the potential for slow or timed release of the biologic molecule in vivo as nanocochleates slowly unwind or otherwise dissociate.

4)       They have a lipid bilayer matrix which serves as a carrier and is composed of simple lipids which are found in animal and plant cell membranes, so that the lipids are non-toxic, non-immunogenic and non-inflammatory.

5)         They are produced easily and safely. (12)

6)       By the use of nanocochleate IV Drugs to be administered orally (e.g. Amphotericin B, a potent antifungal).

7)       They improve oral bioavailability of a broad spectrum of compounds, such as those with poor water solubility, and protein and peptide biopharmaceuticals, which have been difficult to administer. (e.g. ibuprofen for arthritis). (9)

8)       They reduce toxicity stomach irritation and other side effects of the encapsulated drug.

9)       They encapsulate or entrap the subject drug within a crystal matrix rather than chemically bonding with the drug.

10)    They provide protection from degradation to the encochleated drug caused by exposure to adverse environmental conditions such as sunlight, oxygen, water and temperature. (13)

 

Limitations:

1)       They require specific storage condition.

2)       Sometimes aggregation may occur during storage; this can be avoided by the use of aggregation inhibitor.

3)       The cost of manufacturing is high. (7,12)

 

Mechanism of Nanocochleate Drug Delivery:

·         Absorption After oral administration nanocochleates Absorption take place from intestine. Nanocochleates cross across the digestive epithelium and deliver their cargo molecules into blood vessel (Fig. 3).

·         In case of other route except intravenous they cross across the associated cell (in similar manner as discussed above) and reach into circulation.After reaching into circulation they are delivered to targeted cell.

 

Fig. 3: Nanocochleate Absorption from Intestine

 

Delivery to Targeted Cells:

The interaction of calcium with negatively charged lipids has been extensively studied.  Many naturally occurring membrane fusion events involve the interaction of calcium with negatively charged phospholipids (generally phosphatidylserine or phosphatidylglycerol). Calcium-induced perturbations  of  membranes  containing negatively  charged  lipids,  and  the subsequent  membrane  fusion  events,  are important  mechanisms  in  many  natural membrane-fusion  processes.  Hence, cochleates  can  be  envisioned  as membrane-fusion intermediates. (9)

 

Delivery after phagocytosis:-

Macrophages and neutrophils contain membrane phosphatidylserine (PS) receptors which phagocytose nanocochleate.

 

Nanocochleate then comes into close approximation to a liposome membrane, a perturbation and reordering of the liposome membrane is induced, resulting in a fusion event between the outer layer of the nanocochleate and the liposome membrane. This fusion results in the delivery of a small amount of the encochleated material into the cytoplasm of the target cell (Fig. 4). (9)

 

Fig.4: Nanocochleate Delivery to Macrophage

 

Fig. 5: Direct Membrane Fusion

Delivery by cell membrane fusion:-

In such cases nanocochleate first comes into close approximation to a natural membrane, a perturbation and reordering of the cell membrane is induced, resulting in a fusion event between the outer layer of the nanocochleate and the cell membrane. This fusion results in the delivery of a small amount of the encochleated material into the cytoplasm of the target cell. The nanocochleate may slowly fuse or break free of the cell and be available for another fusion event, either with this or another cell     (Fig. 5). (9)

 

Formulation Methods:

The various lipids which are used for the preparation of  nanocochleate  are  phosphotidyl  serine  (PS),  dio -leoylphosphatidylserine  (DOPS),  phosphatidic  acid (PA),  phosphatidylinositol  (PI),  phosphatidyl  glyc -erol  (PG)  and  /or  a  mixture  of  one  or  more  of  these lipids with other lipids. Additionally or alternatively, the lipid can include phosphatidylcholine (PC), phos-phatidylethanolamine  (PE),  diphosphotidylglyc-erol  (DPG),  dioleoyl  phosphatidic  acid  (DOPA),  di-stearoyl phosphatidylserine (DSPS), and dimyristoyl phosphatidylserine  (DMPS),  dipalmitoyl  phosphati -dylgycerol  (DPPG)  . A  multivalent  cation,  which  can be Zn +2or Ca +2 or Mg +2 or Ba +2  and a drug, which can be  protein,  peptide,  polynucleotide,  antiviral  agent, anesthetic,  anticancer  agent,  immunosuppressant, steroidal  anti  inflammatory  agent,  non  steroidal  anti inflammatory  agents,  tranquilizer,  nutritional  sup-plement,  herbal  product,  vitamin  and/or  vasodila-tory agent (14). (Fig 6)

 

Nanocochleates  are  derived  from liposomes  which  are  suspended  in  an aqueous  two-phase  polymer  solution, enabling  the  differential  partitioning  of polar molecule based-structures by phase separation. The liposome-containing two-phase  polymer  solution,  treated  with positively  charged  molecules  such  as  Ca++ or  Zn++,  forms  a  naocochleate  precipitate of a particle size less than one micron. The process  may  be  used  to  produce nanocochleates  containing  biologically relevant molecules(15).

 

·         Method 1 (Hydrogel Method),

·         Method 2 (Trapping method),

·         Method 3(Liposomes before cochleates (LC) dialysis method),

·         Method 4 (Direct calcium (DC) dialysis method),

·         Method 5 (Binary aqueous-aqueous emulsion system).

 

Method 1 (Hydrogel Method):-

This method comprises of following steps:

Step1-  A  suspension  of  small unilamellar  liposomes  or  biologically relevant  molecule-loaded  liposomes  is preparing.  This  can  be  achieved  by standard  methods  such  as  sonication  or microfluidization  or  other  related methods.

Step 2- The liposome suspension is mix with  polymer  A  such  as  dextran  (mol  wt-200,000-500,000),  Polyethylene  glycol (mol  wt-  3400-8000)  or Phosphatidylserine.

Step3-  Preferably  by  injection,  the liposome/Polymer  A  suspension  is  added into another polymer  B such as poly vinyl pyrolidone,  poly  vinyl  alcohol,  ficoll  (mol wt- 30,000- 50,000), and poly vinyl methyl ether (PVMB) (mol wt- 60,000- 160,000) in which  polymer  A  is  nonmiscible,  leading to  an  aqueous  two-phase  system  of polymers.  This  can  be  achieved mechanically  by  using  a  syringe  pump  at an  appropriate  controlled  rate,  for example  a  rate  of  0.1  ml/min  to  50 ml/min, and preferably at a rate of 1 to 10 ml/min.

Step4-   A  solution  of  cation  salt  is added to the two-phase system of step 3, such that the cation diffuses into polymer B and then into the particles comprised of liposome/polymer  A  allowing  the formation of small-sized cochleates.

Step5-  Now  to  isolate  the  cochleate structures  and  to  remove  the  polymer solution,  cochleate  precipitates  are repeatedly  washed  with  a  buffer containing  a  positively  charged  molecule, and  more  preferably,  a  divalent  cation. Addition  of  a  positively  charged  molecule to  the  wash  buffer  ensures  that  the cochleate  structures  are  maintained throughout  the  wash  step,  and  that  they remain as precipitates.Fig-7. (16)

 

 

Fig 6-Cochleate formation from negatively charged vesicles, which fuse after the addition of cation to lead to fused liposomes which roll up to give cochleate cylinders.

 

Fig 7-hydrogel method

 

Method 2 (Trapping method)

This method involves the formation of phosphatidyl-serine liposomes followed by dropwise addition of a solution of  CaCl 2.   Liposomes  can  be  generated  by  either addition of water to phospholipid powder or by adding the water phase to a phospholipid film. A schematic presentation of  the  preparation  methodology is given in  Fig.8. (17)

 

Fig 8-Schematic presentation of trapping method

 

Method 3:  Liposomes before cochleates (LC) dialysis method:-

A  second  method  for  preparing  the small-sized  cochleates  comprises detergent  and  a  biologically  relevant molecule  and  cation.  The detergent is added to disrupt the liposomes.The method comprises the following steps:

Step1-  An  aqueous  suspension  containing  a  detergent-lipid  mixture  is prepared. 

Step2-  The  detergent-lipid  suspension  is  mixed  with  polymer  A  such  as  dextran (mol  wt-200,000-500,000),  Polyethylene  glycol  (mol  wt-  3400-8000)  or  Phosphatidylserine.

Step3-  The  detergent-lipid/polymer  A  suspension  is  added  into  a  solution  comprising  polymer  B  such  as  poly  vinyl  pyrolidone,  poly  vinyl  alcohol,  ficoll  (mol  wt- 30,000- 50,000), and poly vinyl methyl  ether  (PVMB)  (mol  wt-  60,000-  160,000), wherein  polymer  A  and  polymer  B  are immiscible,  thereby  creating  a  two-phase polymer system.

Step4- A solution of a cationic moiety is added to the two-phase polymer system.

Step5-Now  wash  the  two-phase polymer system to remove the polymer. (18)

 

Method 4  (Direct  calcium  (DC)  dialysis method):-

Unlike LC method this method dose not involves the intermediate liposome formation and the cochleates formed were large in size. The mixture of lipid and detergent was  directly  dialyzed  against calcium chloride solution. In this method a competition  between  the  removal  of detergent  from  the  detergent/lipid/drug micelles  and  the  condensation  of  bilayers by calcium, results in needle shaped large dimensional structures. (19)

Step1-  Mixture  of  phosphatidylserine and cholesterol (9:1 wt ratio) in extraction buffer and non‐ionic detergent was mixed with  a  preselected  concentration  of polynucleotide  and  the  solution  was vortexed for 5 minutes.

Step2-  The  clear,  colorless  solution which  resulted  was  dialyzed  at  room temperature  against  three  changes  of  buffer.

Step3-  The  final  dialysis  used  is  6  mM Ca2+,  and  buffer  concentrations  are maintained  compatible  to  cochleate formation.  The resulting white calcium‐phospholipid precipitates have been termed DC cochleates. (18)

 

Method 5 (Binary aqueous-aqueous emulsion system):-

In  this  method  small  liposomes  were  formed  by either  high  pH  or  by  film  method,  and  then  the liposomes are mixed with a polymer, such as dextran. The  dextran/liposome  phase  is  then  injected  into a  second,  non-miscible,  polymer  (i.e.  PEG).  The calcium  was  then  added  and  diffused  slowly  from one  phase  to  another  forming  nanocochleates,  after which  the  gel  is  washed  out.  The  nanocochleates proved  to  promote  oral  delivery  of  injectable  drugs. By this method the  cochleates  formed  are  of  particle size less than 1000 nm. (19)

 

Uses

Nanocochleate is administered to treat at least one disease  or  disorder  selected from the group consisting of, given in table 2

 

Table no 2-Uses

Inflammation  pain

Infection

An immune disorder

genetic  disorders

Cancer

Obesity

Depression

hypertension

Parkinson's  disease

cell  proliferative disorders

Alzheimer's  disease

Schizophrenia

Blood   coagulation  disorders

Grave's  disease

Eczema

Hyperlipidemia

Hyperglycemia

Muscular dystrophy

Arthritis

Asthma

Chronic   rhinosinusitis

Inflammatory  bowel  diseases

Ulcerative

Colitis, and

Crohn's disease

 

Application:-

1)       Development of a  Nanocochleate  based  ApoA1 Formulation for the Treatment of Atherosclerosis and other Coronary Heart Diseases.

2)       Cochleates for the Delivery of Anti-inflammatory Agents.

3)       Cochleates  for  the  Delivery  of  Antimicrobial Agents.

4)       Biogeode  Nanocochleates  have  the  ability to  stabilize  and  protect  an  extended  range  of micronutrients  and  the  potential  to  increase  the nutritional value of processed foods.

5)       Nanocochleates have been used to deliver proteins, peptides  and  DNA  for  vaccine  and  gene  therapy applications.

6)       Nanocochleates  can  deliver  Omega-3  fatty  acids to  cakes,  muffins,  pasta,  soups  and  cookies  without altering the product’s taste or odor .

 

Stability of Nanocochleate:-

These  are  stable,  lipid  based  delivery  formulations whose  structure  and  properties  are  very  different from  liposomes.  This unique structure provides protection from degradation for encochleated, namely, encapsulated molecules. Because the entire cochleate structure  is  a  series  of  solid  layers,components within  the  interior  of  the  cochleate  structure  remain intact,  even  though  its  outer  layers  may  be  exposed to  harsh  environmental  conditions  or  enzymes,  such as in the stomach. Animal studies demonstrated nanocochleates  cross  the  digestive  epithelium  and deliver  their  cargo  molecules  into  target  cells  (20,21).  These  unique  properties  of  nanocochleates  were used  to  mediate  and  enhance  the  oral  bioavailability of  a  broad  spectrum  of  biopharmaceuticals, including  compounds  with  poor  water  solubility, such  as  amphotericin  B   (22,23)..  They  are  stable  to lyophilization and  can  be  reconstituted  with  liquid prior  to  administration.  Cochleate  is  most  versatile technology  for  the  delivery  of  a  wide  range  of  drugs and fragile molecules such as proteins and peptides .

 

CONCLUSION:-

Nanocochleates  are  a  lipid-based  drug  delivery system  that  shows  potential  in  the  oral  delivery of    many  drugs. Nanocochleate  delivery  vehicles  have  been shown  to  be  broadly  applicable  to  a  wide range  of  biologically  important molecules. Since cochleates are formed by ionic interaction between negatively charged  liposomes  and  bivalent cations, microencapsulation  of cationic drugs using themselves as the bridging agents of cochleation can be rationalized. Hence this hydrophilic and multi‐cationic drugs and peptides can be successfully encapsulated into cochleates  and  nanocochleate  particles  as  cochleates’  bridging agent. These  unique  properties  enable cochleates  and  nanocochleates  to  deliver  charged hydrophilic  drugs  across  the  tissue  membrane.  Thus nanochleate possesses  strong  therapeutic  potential in the area of drug delivery.

 

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Received on 17.01.2012

Accepted on 08.04.2012        

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 4(3): May-June 2012, 153-159