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Resealed Erythrocytes: As a Novel Drug Delivery Carrier
Submitted by ayushsinghal on Thu, 09/10/2009 - 20:51

Erythrocytes (RBCs) have potential carrier capabilities for the delivery of drugs. Erythrocytes are biocompatible, biodegradable, possess long circulation half
lives, and can be loaded with a variety of biologically active compounds using various chemical and physical methods.
Erythrocytes have been extensively studied for their potential carrier capabilities for the delivery of drugs and drug-loaded microspheres. Such drug-loaded
carrier erythrocytes are simply prepared by collecting blood samples from the interested organism, separating erythrocytes from plasma, entrapping drug in
erythrocytes, and resealing the resultant cellular carriers. Hence, these carriers are called resealed erythrocytes. The overall process is based on the response of
these cells under osmotic conditions. Upon reinjection, the drug-loaded erythrocytes serve as slow circulating depots and target the drugs to a
reticuloendothelial system (RES).
Resealed erythrocytes have applications in fields of human and veterinary medicine.
>Such cells could be used as circulating carriers to disseminate a drug within a prolonged period of time in circulation or in target-specific organs, including the
liver, spleen, and lymph nodes.
>A majority of the drug delivery studies using drug-loaded erythrocytes are in the preclinical phase. Antineoplastic drugs such as methotrexate, bleomycin,
asparginase and adriamycin have been successfully delivered by erythrocytes.
>Removal of RES iron overload, Removal of toxic agents and Delivery of antiviral agents are some other applications of resealed erythrocytes.
Biopharmaceuticals, therapeutically significant peptides and proteins, nucleic acid-based biologicals, antigens and vaccines, are among the recently focused
pharmaceuticals for being delivered using carrier erythrocytes.
Click to see next slide
Thanks Mr. Amol for your comments..
Submitted by ayushsinghal on Tue, 09/15/2009 - 10:54.
Regarding drug loading and content uniformity:
1)All the techniques of drug loading mentioned in slide-12 and explanations in slides 13-16 are sufficient for the drug loading purpose and the red cell loader is
currently in use for the same.
2)Regarding content uniformity almost all erythrocytes have similar dimensional characteristics so there is very less chance of possibility of non uniform drug
loading.One possibility serves is the presence of immature erythrocytes in the sample to be resealed but for that the collected samples are pre-characterized for
whether they are opt for resealing or not and the techniques used for that are already mentioned in slide no 6.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
http://www.pharmainfo.net/ayushsinghal/biography
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Hello Ayush!
Submitted by rujuta_mehendale on Sun, 09/13/2009 - 14:39.
You have selected a very good topic. Congratulations ! I think it is a very promising technology and it would really help if there is some research work done on
'sealed erythrocytes'. All the best..
My question to you is what is the compatibility parameter of the RBCs?? Is it compatible with all the drugs that are formulated(anti- virals, antiparasitics or
even hepatic application based drugs) using this technology?
Thanks!
Thanks Miss.Rujuta for the comments...
The answers to your questions are as below
1) First of all in general regarding bio-compatibility of resealed erythrocytes
i would like to inform you that the circulation survival kinetics of resealed erythrocytes show typical bimodal behavior with a rapid loss of cells during the first
24 h after injection, followed by a slow decline phase with a half life on the order of days or weeks.The early loss accounts for 15–65% loss of total injected
cells.Thus slow proceeding of therapy can make these cells compatible within the body.
2)Regarding compatibility of the drugs that are being formulated,as you know, using this technology : a majority of the drug delivery studies using drug-loaded
erythrocytes are in the preclinical phase. In a few clinical studies, successful result have been obtained which shows that most of the drugs formulated are
compatible.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
a very nice prsentation,

Submitted by shreeshabhat on Fri, 09/11/2009 - 14:40.
a very nice prsentation, ayush!!
what is the potential of resealed erythrocytes in india? and wont there be any problem in its storage and its export to different countries?
Shreesha V Bhat
Ramanbhai Patel College of Pharmacy, Education campus Changa,
Gujarat, India.
http://www.pharmainfo.net/shreeshabhat
A very nice and interesting question!!

Submitted by ayushsinghal on Sat, 09/12/2009 - 11:29.
Answers to your questions are as below:
1)Regarding potential of resealed erythrocytes in India:
This drug delivery option is a very potent and is target specific too but it is not an economic process.So in a way less potential for a developing country like
India.
But a positive approach says that once established we can find some economic way out so that this means of drug delivery can be well established in our
Country too.
2)Regarding in-vitro storage and transport:
The lack of reliable and practical storage methods has been a limiting factor for the wide-spread clinical use of the carrier erythrocytes.
---The most common storage media include Hank’s balanced salt solution and acid–citrate–dextrose at 4 degree Celsius. Cells remain viable in terms of their
physiologic and carrier characteristics for at least 2 weeks at this temperature.
---The addition of calcium-chelating agents or the purine nucleosides improve circulation survival time of cells upon re-injection.
Storage can be done same way and transported via fastest means after the order is registered but this may again add to the cost and make it uneconomic for a an
average human to go for.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
hiiiiiiiiiiii
Submitted by Priyanka bhosale on Thu, 03/31/2011 - 14:42.
sir ur presentation is nice i like it. i want this ppt so plz can u mail me all this ppt on my email id priyanka.bhosale10@gmail.com
p.p.bhosale
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Thanks for your comments Mr.Jithan.......
Submitted by ayushsinghal on Wed, 09/23/2009 - 15:48.
Regarding assuring the stability of products made out of resealed erythrocytes...

First i want to inform you about stability parameters...
1) Since the resealed products are copies similar to our own blood erythrocytes they posses a good in vivo stability parameter.
2)Moreover the addition of calcium-chelating agents or the purine nucleosides improve circulation survival time of cells upon re-injection.
Next your query regarding assuring stability...
There a some special techniques regarding assuring the stability of resealed products..
1) use of gamma-ray perturbed angular correlation (PAC) techniques.
2) standard in vitro leakage methods employing sequestered labeled markers.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Rbcs count
Submitted by jigardpatel on Thu, 09/17/2009 - 09:01.
Hello Ayush;
Really a good topic selection;
Is the resealed erythrocytes interfer in the rbcs count in the blood and is there any chances of occuring polycythemia????
Thanks Mr.Jigar for the comments....

A good question....
Regarding interference of resealed erythrocytes i wish to clear two major points that are quite important in context to your question:
1)We are using resealed erythrocytes with a view to deliver drugs so naturally a huge amount of cells are neither needed nor can be transfused at a time into the
patients systemic circulation, if so there are 100% changes of severe toxicity.
2) In primary polycythemia, there may be 8 to 9 million and occasionally 11 million erythrocytes per cubic millimeter of blood(a normal range for adults is 4-
6).
So from the above two points you may have got it that resealed erythrocytes can neither interfere with normal blood RBC count nor will they lead to
polycthemia.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
good presentation
Submitted by nirajvyas4me on Tue, 09/15/2009 - 10:22.
hi ayush
i want to ask a question..
my question is that
can you give me any company name which is corrently working or manufacturing any product related to resealed erythrocytes mechanism.???
bye
Thanks for your comments sir...

A very interesting question...

A majority of the drug delivery studies using drug-loaded erythrocytes are in the preclinical phase.There is no such open reference of any company working on
the resealed erythrocytes.
But many Universities as below are involved in research on same:-
College station & Texas in USA;
Institute of Biochemistry, University of Urbino, Italy;
Laboratory of Virology, Istituto Supanita, Rome, Italy;
§Lepetit Research Center,Italy ;
Institute of Biochemistry, University Genoa,Genoa, Italy.
and many others...
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
drug loading and content uniformity

Submitted by amolsmalpani on Sun, 09/13/2009 - 18:50.
Dear Ayush,
resealed erythrocytes is having potential but what about drug loading and content uniformity? Suggest some remedies.
Regards,
Amol
Amol S Malpani
Second prize Winners of Skills Test 2010


Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Hello Ayush!
Thanks!


compatible.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT

Shreesha V Bhat
Gujarat, India.


India.
Country too.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
hiiiiiiiiiiii
p.p.bhosale
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Comments
It is good
Submitted by kiran_mandlem on Wed, 10/14/2009 - 09:08.
IS it possible to delivery the antibodies that target the cancer through resealed erythrocytes. If so what are the barriers.
Kiran
My Page Link : http://www.pharmainfo.net/kiranmandlem
Thanks for your comments Sir.......

Antineoplastic drugs such as methotrexate, bleomycin, asparginase, and adriamycin have been successfully delivered by erythrocytes. Agents such as
daunorubicin diffuse rapidly from the cells upon loading and hence pose a problem. This problem can be overcome by covalently linking daunorubicin on the
erythrocytic membrane using gluteraldehyde or cis-aconitic acid as a spacer. The resealed erythrocytes loaded with carboplatin show localization in liver.
Since this medium of delivery is prominently based upon transport of drug by on linking with membrane no such barriers to this have been reported.
thank you.....
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Mr. Singhal, Do the effects

Submitted by Aarti Anantram on Sun, 10/04/2009 - 18:23.
Mr. Singhal,
Do the effects of drug incorporated into erythrocytes persist for the entire lifespan of the erythrocyte?
Regards,
Aarti
Thanks for your comments .......

Submitted by ayushsinghal on Mon, 10/05/2009 - 13:34.
Life span of the resealed erythrocytes is of no importance since the drug release mechanism is by destruction or metabolism of these cells by the RES system of
the body.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
good
Submitted by ankitsheelamittal on Sun, 10/04/2009 - 15:40.
hi.
Ayush A. Singhal
can it will harmful for person having GIT distubances ?
well pls tellme abt the cost effectiveness ?
also tell me the compatibility of same?
Mention some QA/QC test for the same ?
thanks
1)No there is no harm to the person having GIT disturbance since the drug has no relation with GIT based absorption.
2)Cost may be higher at a first instance but a proper set up and planning can bring it down effectively.
3)They have a very high compatibility since body's immune system cannot differentiate between normal and resealed erythrocytes.
4)Pre-characterization parameters are already mentioned which will ensure quality of resealed erythrocytes.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
nice designed presentation

Submitted by ankit on Sun, 10/04/2009 - 14:38.
Dear Ayush,
very well designed presentation.
please give me such idea about the biodigradation process of
Resealed Erythrocytes & by what?
is there any significance regarding biodigradate product of it?
regard
ankit
http://www.pharmainfo.net/ankit

Old damaged RBC's are removed from the circulation in the following ways:
1. 90% are removed from the circulation by the phagocytic activities of macrophages in the liver, spleen and lymph nodes.(RES)
2. 10% of the old cells hemolyze in the circulation. The fragments of these cells are then engulfed by macrophages.
3. The chemical components of the RBC are broken down within vacuoles of the macrophages due to the action of lysosomal enzymes.
The same way resealed being modified erythrocytes are prone to RES activity and the products released are the drug loaded to them.So the biodegradate
product is of utter importance.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Resealed Erythrocytes
Submitted by chitrakaladhuri on Sat, 10/03/2009 - 01:58.
Its a good presentation & topic.

Your characterization slide was difficult to read...can you please tell me what is the need to study characterization parameters.
This technique involves highly specialized methods of preparation, dont you think this will make this system highly expensive & time consuming..& there is
always a danger of its purity, if at any stage any thing goes wrong it may eventually show adverse effects. Comment
With the continuous use of these in cases like cancer, or such disease where prolong & repetitive medication is required, what the chances of inducing mutation.
As treated erythrocytes are used, what if mutation of the erythrocytes takes place. Comment
Thank you

1)Such problems are due to compression problems during slide uploading.characterization studies are of utmost importance because such parameters are to be
noted during resealing so that mature erythrocytes having similar drug loading capacity can be choosen.
2)No..This is not too expensive and time consuming as you think but needs correct methodology of set up by technicians.
for eg.a red cell loader is fastest way of resealing.though may look costly to set up once but later it can serve to many patients and that will surely tremendously
reduce the cost of same.
3)No.Still there is no such evidence of mutation yet reported.
---We are still facing with the problem of drug resistance with long term therapy so resealed and drug resistance have no connection in context to your
question.If such a problem exists with normal therapy there are chances they may develop in this case too.
---Resealed are means of fast and better drug delivery not fighting mutations.But since they maintain an optimum therapeutic level in patients body constantly
they can be searched in a future for fighting drug resistance due to mutations too.
thank you......
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Nice and very imprsessive Presentation!!!

Submitted by apurva.anugamini on Fri, 10/02/2009 - 14:33.
hello,
What do you mean by heparinised tube?

Submitted by ayushsinghal on Fri, 10/02/2009 - 14:38.
--------------Heparin Tube is widely used in blood collection and anti-coagulation and for clinical biochemistry, emergency biochemistry tests and also for some
hemorrheology.It is used for plasma determinations in chemistry.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Hello Ayush
Submitted by nikdream on Fri, 10/02/2009 - 14:16.
Can you explain detail about electro encapsulation method of drug loading? is there any disadvantage?
Nikhil

1) "Electro-insertion or electroencapsulation":-An electrical pulse method to encapsulate bioactive molecules.Also known as electroporation, the method is
based on the observation that electrical shock brings about irreversible changes in an erythrocyte membrane.The erythrocyte membrane is opened by a
dielectric breakdown.Subsequently, the pores can be resealed by incubation at 37 *C in an isotonic medium.
2) But main drawbacks are the need for special instrumentation and the sophistication of the process and the entrapment efficiency of this method is about 35%.
thank you.....
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
interaction
Submitted by ganesh.nirma on Fri, 10/02/2009 - 13:34.
hi,
Nice presentation.
I want to ask That will body consider them as same When they are introduced again after resealing and it will follow the same cycle of RBC metabolism??
Regards
Ganesh.
1)No.The body or immune system can not differentiate between normal and resealed erythrocytes because of similar characteristic properties as normal ones .
2)Yes.They will follow same cycle of RBC metabolism by RES cells and that is the mechanism behind release of drugs by drug loaded erythrocytes.
thank you....
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Overall nice presentation; Is

Submitted by Khushbu Hasmukh... on Fri, 10/02/2009 - 13:26.
Overall nice presentation;

Is therre any side effect of resealed erythrocyte on blood rbcs count???
give the name marketed preparation of this drug delivery system?
Thank you.
K.H.Patel.
My profile link is
http://www.pharmainfo.net/khushbu-hasmukh-patel
patients systemic circulation,so no effect on RBC count.
2)Marketed products currently under study:
----antiviral drug i.,e AZEDOTHYMIDINE.
----antimyobacterial drug i.,e ETHAMBUTOL.
AZTpEMB , AZTpEMBpAZT , AZTp2EMB are some of the compounds, synthesized which contain anti-retrovial and antimicrobial activity are used in the
trearment of M.AVIUM infection and in bacterial infections of AIDS.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
DEAR AYUSH
Submitted by kunal_ingale on Wed, 09/30/2009 - 11:49.
nice presentation
I want to ask is there any marketed preparation of this technology?
While collecting blood sample is it needed to keep the blood group report,
Is there any interaction of blood group while doing such preparation?

Answers to your questions:-

2)Regarding blood group since agglutinins are present on walls of erythrocytes there are chances of antigen antibody reactions so there are 2 options:
--- modification of erythrocytes membrane.
--- checking blood group and compatibility parameters.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
great work
Submitted by Hrushikesh Kara... on Tue, 09/29/2009 - 07:57.
Sir, its really great work

What is the stability profile of Resealed erytrocytes??
Another very comic question.. Can we make available these resealed erythrocytes in BLOOD BANK?
thanks
Hrushikesh
Thanks for your comments Mr.Hrushikesh.......

Answers to your questions....

a) about stability parameters...
b)NO we cant make resealed erythrocytes available in blood banks because of :-
---The lack of reliable and practical storage methods .
thank you....
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
GOLDEN EGGS OF NDDS..

Submitted by sirisha on Thu, 09/24/2009 - 16:00.
dear ayush..
Very good presentation from your side..
Resealed Erythrocytes is very innovative topic..Coming to the queries,,
1. Does these erythrocytes also possess the same lifespan like original cells??
2. When these resealed erythrocytes are introduced into body..do they trigger immune system..?
3. For encapsulating drug within erythrocyte ghosts..can electroporation technique be used?
Sirisha Pingali
http://www.pharmainfo.net/sirisha/biography
Thanks for your comments Miss Sirisha.......
Answers to your queries.....

1)No.
-In a storage media like Hank’s balanced salt solution and acid–citrate–dextrose at 4*C.Cells remain viable in terms of their physiologic and carrier
characteristics for at least 2 weeks at this temperature.
-In vivo life span is not of much importance since we aim at drug release by means of phagoctosis of these by RES.
2)No on introduction into the body's systemic circulation these do not trigger an immune response.
--Since autologous cells are used, there is no possibility of triggered immune response
3)Yes electroporation can be used...
--------"Electro-insertion or electroencapsulation":-An electrical pulse method to encapsulate bioactive molecules.Also known as electroporation, the method is
-----------But main drawbacks are the need for special instrumentation and the sophistication of the process and the entrapment efficiency of this method is
about 35%.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
dear ayush.. Good
Submitted by sirisha on Fri, 09/25/2009 - 16:11.
dear ayush..
Good explanation..
Sirisha Pingali
Nice presentation
Submitted by drgunasakaran1 on Wed, 09/23/2009 - 17:05.
Dear Ayush,
Nice presentation, kindly share with us, what are all the drugs currently under research using resealed erthyocytes as drug delivery carrier.
Dr.S.Gunasakaran,MBBS,MD.
Currently a wide range of drugs and enzymes have been delivered or are under research as proposed...A few examples of these are as listed below:--
--Antineoplastic drugs such as methotrexate, bleomycin, asparginase, and
adriamycin have been successfully delivered by erythrocytes.
--Resealed erythrocytes have been used to deliver deoxycytidine derivatives, recombinant herpes simplex virus type 1 (HSV-1) glycoprotein B, azidothymidine
derivatives, aza-thioprene, acyclovir, and fludarabine phosphate.
--The enzymes used include B-glucosidase, B-glucoronidase, B-galactosidase.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
queries...
Submitted by Lakshmi on Wed, 09/23/2009 - 15:33.
Hello Ayush A.Singhal,
Could you please clarify the following points:
1. Regarding the advantages of resealed erythrocytes you have mentioned about bio compatibility where these resealed erythrocytes are not prone to the action
of immune system. But phagocytosis which is an important mechanism of drug release is due to an immune response. Could you please explain these
contraversial statements of yours?
2. As you have mentioned in the presentation and also in the reply to one of the query posted by co participants, comparitively very small amount of resealed
erythrocytes are introduced into systemic circulation and their distribution is random. Does it mean that the amount of drug released per day is a chance factor
but not an ensured rate?
3. Regarding the slides entitled as "Hypnotic dialysis" and "Hypnotic preswelling" Was that an unnoticed error of yours or is there any meaningful method of
such type? In case its a mistake I suggest you to take care in future presentations as the tittle is a crucial part of slides.
thanks for your comment Miss. Indira....

Clarifications a word worth while as per your questions...

1) You are just not able to differentiate/get what the content need to say.Anyways phagocytosis by RES cells is the termination point for most of the cells and
for the resealed erythrocytes too at this point the drug will be released.
Damaged erythrocytes are rapidly cleared from circulation by phagocytic Kupffer cells in liver and spleen.Resealed erythrocytes, by modifying their
membranes, can there-fore be used to target the liver and spleen.
Phagoctosis here is due to RES organs which play an imp. role in termination of old and damaged cells and not an immune responce.
2) Not at all.The reason for this:
a) If sustained released is needed your question does not apply since equal distribution of cells throughout blood will cause only particular amt of cells being
phagoctosised by RES cell in particular interval of time.
b) although for delivery to target-specific RES organs, rapid phagocytosis
and hence a shorter life span is desirable.So all the infused resealed cells will undergo phagoctosis and requisite conc. will be achieved in particular interval of
time.
3)I am really sorry for that. It is a typing error as i had to design whole presentation in a very less period of time..But i think this is worth ignoring..I have
already sent a new rectified copy to the authority but due to late retrieval they said that it was not possible to change the content now...
It is my generous request to all the viewers to take note of following typing errors:
1)Hypotonic dialysis not Hypnotic dialysis
2)Hypotonic preswelling not Hypnotic preswelling
Elsewhile eyerything is perfect......
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
but...........
Submitted by Lakshmi on Sun, 10/04/2009 - 13:38.
I think you dint understand my second question... In your answer do you mean to say that the body differentiates the resealed erythrocytes from normal RBCs
and specifically phagocytizes them after administration? If no, then there should be a random phenomena.. That was my question. Kindly give me a clear
explaination..
Erythrocytes!!
Submitted by aukunjv on Wed, 09/23/2009 - 13:45.
Hi,
Good presentation. Can you assure the stability of products made out of resealed erythrocytes?
Jithan


Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Rbcs count
Hello Ayush;

A good question....
6).
polycthemia.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
good presentation
hi ayush
my question is that
bye


and many others...
Ayush A. Singhal
RPCP, CHANGA
GUJARAT

Dear Ayush,
Regards,
Amol
Amol S Malpani


Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Hello Ayush!
Thanks!


compatible.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT


Shreesha V Bhat
Gujarat, India.

India.
Country too.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
hiiiiiiiiiiii
p.p.bhosale
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Comments
It is good
Submitted by kiran_mandlem on Wed, 10/14/2009 - 09:08.
IS it possible to delivery the antibodies that target the cancer through resealed erythrocytes. If so what are the barriers.
Kiran
My Page Link : http://www.pharmainfo.net/kiranmandlem

Antineoplastic drugs such as methotrexate, bleomycin, asparginase, and adriamycin have been successfully delivered by erythrocytes. Agents such as
daunorubicin diffuse rapidly from the cells upon loading and hence pose a problem. This problem can be overcome by covalently linking daunorubicin on the
erythrocytic membrane using gluteraldehyde or cis-aconitic acid as a spacer. The resealed erythrocytes loaded with carboplatin show localization in liver.
Since this medium of delivery is prominently based upon transport of drug by on linking with membrane no such barriers to this have been reported.
thank you.....
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Mr. Singhal, Do the effects

Submitted by Aarti Anantram on Sun, 10/04/2009 - 18:23.
Mr. Singhal,
Do the effects of drug incorporated into erythrocytes persist for the entire lifespan of the erythrocyte?
Regards,
Aarti

Life span of the resealed erythrocytes is of no importance since the drug release mechanism is by destruction or metabolism of these cells by the RES system of
the body.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
good
Submitted by ankitsheelamittal on Sun, 10/04/2009 - 15:40.
hi.
Ayush A. Singhal
can it will harmful for person having GIT distubances ?
well pls tellme abt the cost effectiveness ?
also tell me the compatibility of same?
Mention some QA/QC test for the same ?
thanks
1)No there is no harm to the person having GIT disturbance since the drug has no relation with GIT based absorption.
2)Cost may be higher at a first instance but a proper set up and planning can bring it down effectively.
3)They have a very high compatibility since body's immune system cannot differentiate between normal and resealed erythrocytes.
4)Pre-characterization parameters are already mentioned which will ensure quality of resealed erythrocytes.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
nice designed presentation

Submitted by ankit on Sun, 10/04/2009 - 14:38.
Dear Ayush,
very well designed presentation.
please give me such idea about the biodigradation process of
Resealed Erythrocytes & by what?
is there any significance regarding biodigradate product of it?
regard
ankit
http://www.pharmainfo.net/ankit

Old damaged RBC's are removed from the circulation in the following ways:
1. 90% are removed from the circulation by the phagocytic activities of macrophages in the liver, spleen and lymph nodes.(RES)
2. 10% of the old cells hemolyze in the circulation. The fragments of these cells are then engulfed by macrophages.
3. The chemical components of the RBC are broken down within vacuoles of the macrophages due to the action of lysosomal enzymes.
The same way resealed being modified erythrocytes are prone to RES activity and the products released are the drug loaded to them.So the biodegradate
product is of utter importance.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Resealed Erythrocytes
Submitted by chitrakaladhuri on Sat, 10/03/2009 - 01:58.
Its a good presentation & topic.

Your characterization slide was difficult to read...can you please tell me what is the need to study characterization parameters.
This technique involves highly specialized methods of preparation, dont you think this will make this system highly expensive & time consuming..& there is
always a danger of its purity, if at any stage any thing goes wrong it may eventually show adverse effects. Comment
With the continuous use of these in cases like cancer, or such disease where prolong & repetitive medication is required, what the chances of inducing mutation.
As treated erythrocytes are used, what if mutation of the erythrocytes takes place. Comment
Thank you

1)Such problems are due to compression problems during slide uploading.characterization studies are of utmost importance because such parameters are to be
noted during resealing so that mature erythrocytes having similar drug loading capacity can be choosen.
2)No..This is not too expensive and time consuming as you think but needs correct methodology of set up by technicians.
for eg.a red cell loader is fastest way of resealing.though may look costly to set up once but later it can serve to many patients and that will surely tremendously
reduce the cost of same.
3)No.Still there is no such evidence of mutation yet reported.
---We are still facing with the problem of drug resistance with long term therapy so resealed and drug resistance have no connection in context to your
question.If such a problem exists with normal therapy there are chances they may develop in this case too.
---Resealed are means of fast and better drug delivery not fighting mutations.But since they maintain an optimum therapeutic level in patients body constantly
they can be searched in a future for fighting drug resistance due to mutations too.
thank you......
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Nice and very imprsessive Presentation!!!

Submitted by apurva.anugamini on Fri, 10/02/2009 - 14:33.
hello,
What do you mean by heparinised tube?

--------------Heparin Tube is widely used in blood collection and anti-coagulation and for clinical biochemistry, emergency biochemistry tests and also for some
hemorrheology.It is used for plasma determinations in chemistry.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Hello Ayush
Submitted by nikdream on Fri, 10/02/2009 - 14:16.
Can you explain detail about electro encapsulation method of drug loading? is there any disadvantage?
Nikhil

1) "Electro-insertion or electroencapsulation":-An electrical pulse method to encapsulate bioactive molecules.Also known as electroporation, the method is
2) But main drawbacks are the need for special instrumentation and the sophistication of the process and the entrapment efficiency of this method is about 35%.
thank you.....
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
interaction
Submitted by ganesh.nirma on Fri, 10/02/2009 - 13:34.
hi,
Nice presentation.
I want to ask That will body consider them as same When they are introduced again after resealing and it will follow the same cycle of RBC metabolism??
Regards
Ganesh.
1)No.The body or immune system can not differentiate between normal and resealed erythrocytes because of similar characteristic properties as normal ones .
2)Yes.They will follow same cycle of RBC metabolism by RES cells and that is the mechanism behind release of drugs by drug loaded erythrocytes.
thank you....
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Overall nice presentation; Is

Submitted by Khushbu Hasmukh... on Fri, 10/02/2009 - 13:26.
Overall nice presentation;

Is therre any side effect of resealed erythrocyte on blood rbcs count???
give the name marketed preparation of this drug delivery system?
Thank you.
K.H.Patel.
My profile link is
http://www.pharmainfo.net/khushbu-hasmukh-patel
patients systemic circulation,so no effect on RBC count.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
DEAR AYUSH
Submitted by kunal_ingale on Wed, 09/30/2009 - 11:49.
nice presentation
I want to ask is there any marketed preparation of this technology?
While collecting blood sample is it needed to keep the blood group report,
Is there any interaction of blood group while doing such preparation?

Answers to your questions:-

2)Regarding blood group since agglutinins are present on walls of erythrocytes there are chances of antigen antibody reactions so there are 2 options:
--- modification of erythrocytes membrane.
--- checking blood group and compatibility parameters.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
great work
Submitted by Hrushikesh Kara... on Tue, 09/29/2009 - 07:57.
Sir, its really great work

What is the stability profile of Resealed erytrocytes??
Another very comic question.. Can we make available these resealed erythrocytes in BLOOD BANK?
thanks
Hrushikesh
Thanks for your comments Mr.Hrushikesh.......

Answers to your questions....

a) about stability parameters...
b)NO we cant make resealed erythrocytes available in blood banks because of :-
---The lack of reliable and practical storage methods .
thank you....
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
GOLDEN EGGS OF NDDS..

Submitted by sirisha on Thu, 09/24/2009 - 16:00.
dear ayush..
Very good presentation from your side..
Resealed Erythrocytes is very innovative topic..Coming to the queries,,
1. Does these erythrocytes also possess the same lifespan like original cells??
2. When these resealed erythrocytes are introduced into body..do they trigger immune system..?
3. For encapsulating drug within erythrocyte ghosts..can electroporation technique be used?
Sirisha Pingali
Thanks for your comments Miss Sirisha.......
Answers to your queries.....

1)No.
-In a storage media like Hank’s balanced salt solution and acid–citrate–dextrose at 4*C.Cells remain viable in terms of their physiologic and carrier
characteristics for at least 2 weeks at this temperature.
-In vivo life span is not of much importance since we aim at drug release by means of phagoctosis of these by RES.
2)No on introduction into the body's systemic circulation these do not trigger an immune response.
--Since autologous cells are used, there is no possibility of triggered immune response
3)Yes electroporation can be used...
--------"Electro-insertion or electroencapsulation":-An electrical pulse method to encapsulate bioactive molecules.Also known as electroporation, the method is
-----------But main drawbacks are the need for special instrumentation and the sophistication of the process and the entrapment efficiency of this method is
about 35%.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
dear ayush.. Good
Submitted by sirisha on Fri, 09/25/2009 - 16:11.
dear ayush..
Good explanation..
Sirisha Pingali
Nice presentation
Submitted by drgunasakaran1 on Wed, 09/23/2009 - 17:05.
Dear Ayush,
Nice presentation, kindly share with us, what are all the drugs currently under research using resealed erthyocytes as drug delivery carrier.
Dr.S.Gunasakaran,MBBS,MD.
Currently a wide range of drugs and enzymes have been delivered or are under research as proposed...A few examples of these are as listed below:--
--Antineoplastic drugs such as methotrexate, bleomycin, asparginase, and
adriamycin have been successfully delivered by erythrocytes.
--Resealed erythrocytes have been used to deliver deoxycytidine derivatives, recombinant herpes simplex virus type 1 (HSV-1) glycoprotein B, azidothymidine
derivatives, aza-thioprene, acyclovir, and fludarabine phosphate.
--The enzymes used include B-glucosidase, B-glucoronidase, B-galactosidase.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
queries...
Submitted by Lakshmi on Wed, 09/23/2009 - 15:33.
Hello Ayush A.Singhal,
Could you please clarify the following points:
1. Regarding the advantages of resealed erythrocytes you have mentioned about bio compatibility where these resealed erythrocytes are not prone to the action
of immune system. But phagocytosis which is an important mechanism of drug release is due to an immune response. Could you please explain these
contraversial statements of yours?
2. As you have mentioned in the presentation and also in the reply to one of the query posted by co participants, comparitively very small amount of resealed
erythrocytes are introduced into systemic circulation and their distribution is random. Does it mean that the amount of drug released per day is a chance factor
but not an ensured rate?
3. Regarding the slides entitled as "Hypnotic dialysis" and "Hypnotic preswelling" Was that an unnoticed error of yours or is there any meaningful method of
such type? In case its a mistake I suggest you to take care in future presentations as the tittle is a crucial part of slides.
thanks for your comment Miss. Indira....

Clarifications a word worth while as per your questions...

1) You are just not able to differentiate/get what the content need to say.Anyways phagocytosis by RES cells is the termination point for most of the cells and
for the resealed erythrocytes too at this point the drug will be released.
Damaged erythrocytes are rapidly cleared from circulation by phagocytic Kupffer cells in liver and spleen.Resealed erythrocytes, by modifying their
membranes, can there-fore be used to target the liver and spleen.
Phagoctosis here is due to RES organs which play an imp. role in termination of old and damaged cells and not an immune responce.
2) Not at all.The reason for this:
a) If sustained released is needed your question does not apply since equal distribution of cells throughout blood will cause only particular amt of cells being
phagoctosised by RES cell in particular interval of time.
b) although for delivery to target-specific RES organs, rapid phagocytosis
and hence a shorter life span is desirable.So all the infused resealed cells will undergo phagoctosis and requisite conc. will be achieved in particular interval of
time.
3)I am really sorry for that. It is a typing error as i had to design whole presentation in a very less period of time..But i think this is worth ignoring..I have
already sent a new rectified copy to the authority but due to late retrieval they said that it was not possible to change the content now...
It is my generous request to all the viewers to take note of following typing errors:
1)Hypotonic dialysis not Hypnotic dialysis
2)Hypotonic preswelling not Hypnotic preswelling
Elsewhile eyerything is perfect......
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
but...........
Submitted by Lakshmi on Sun, 10/04/2009 - 13:38.
I think you dint understand my second question... In your answer do you mean to say that the body differentiates the resealed erythrocytes from normal RBCs
and specifically phagocytizes them after administration? If no, then there should be a random phenomena.. That was my question. Kindly give me a clear
explaination..
Erythrocytes!!
Submitted by aukunjv on Wed, 09/23/2009 - 13:45.
Hi,
Good presentation. Can you assure the stability of products made out of resealed erythrocytes?
Jithan


Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Rbcs count
Hello Ayush;

A good question....
6).
polycthemia.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
good presentation
hi ayush
my question is that
bye


and many others...
Ayush A. Singhal
RPCP, CHANGA
GUJARAT

Dear Ayush,
Regards,
Amol
Amol S Malpani


Ayush A. Singhal
RPCP, CHANGA
GUJARAT
Hello Ayush!
Thanks!


compatible.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT


Shreesha V Bhat
Gujarat, India.

India.
Country too.
Ayush A. Singhal
RPCP, CHANGA
GUJARAT
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June 2002, Volume 9, Number 11, Pages 749-751
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RESEARCH ARTICLE
Year : 2010 | Volume : 4 | Issue : 2 | Page : 116-120
Engineered cellular carrier nanoerythrosomes as potential targeting vectors for anti-malarial drug
X 10
Jaya Agnihotri, Virendra Gajbhiye, Narendra Kumar Jain

Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar 1
(MP), India
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• Agnihotri J
• Gajbhiye V
• Jain NK
Abstract
The present investigation was aimed at developing and exploring the use of nanoerythrosomes (nEs) for targeted delivery
of anti-malarial drug, pyrimethamine (PMA). The formulation was prepared by the extrusion method and drug was
conjugated to nEs with the help of gluteraldehyde used as a cross-linking agent. The nEs formulation was optimized for
drug concentration, surface morphology, viscosity, and sedimentation volume. The drug-loaded nEs showed delayed in
vitro release, good stability at 4±1°C, and controlled in vivo release. Tissue distribution studies showed higher
accumulation of drug in liver (17.34±1.3 μg/ml) at 3 h in the case of nEs as compared to free drug (12.82±0.7 μg/ml). A
higher amount of drug i.e. 13.14±0.9 μg/ml was found after 24 h in liver in the case of nEs as compared to free drug
concentration of 9.72±0.5 μg/ml. Data showed that developed PMA-loaded nEs hold promise for targeting and
controlling the release of drug and improve treatment of malaria.
Keywords: Anti-malarial, cellular carrier, nanoerythrosomes, pyrimethamine, targeting
How to cite this article:
Agnihotri J, Gajbhiye V, Jain NK. Engineered cellular carrier nanoerythrosomes as potential targeting
vectors for anti-malarial drug. Asian J Pharm 2010;4:116-20
How to cite this URL:

Agnihotri J, Gajbhiye V, Jain NK. Engineered cellular carrier nanoerythrosomes as potential targeting
vectors for anti-malarial drug. Asian J Pharm [serial online] 2010 [cited 2011 May 16];4:116-20. Available
from: http://www.asiapharmaceutics.info/text.asp?2010/4/2/116/68462
Introduction
G
a
d
Malaria is the most important disease of humans, affecting 109 endemic countries in year 2008 with a g
population of over 1 billion people and causing around 1 million deaths each year. [1] There has been a e
t
resurgence of this disease in many parts of the tropics not withstanding enormous control efforts. In s
addition, drug resistance causes increasing problems in most malaria-affected areas. [2] Malaria remains
p
today as it has been for centuries, a major burden on tropical communities and a danger to travelers. o
Benign malarias (Plasmodium vivax, P. ovale and P. malariae) are rarely fatal and are still sensitive to w
e
chloroquine in most of the clinical situations. Malignant malaria caused by P. falciparum, ranks high as a r
cause of morbidity and mortality in tropics. [3] P. falciparum infection causes cerebral malaria as a e
d
complication, although complicated malaria can and does involve other tissue and organs in the body. [4]
The P. falciparum malaria may sometimes be managed with chloroquine, although there is now an b
y
increased tendency toward the use of either pyrimethamine (PMA) with quinine or mefloquine. PMA is an
anti-malarial drug which acts by inhibiting the conversion of folic acid to folinic acid. [5] It is widely used in G
o
the treatment of P. falciparum infection along with sulfodixine as combination therapy, but successful o
use of PMA is limited due to its toxic effect, particularly depression of hematopoisis. Acute over dose g
l
causes convulsion, tachycardia, respiratory depression, circulatory collapse, and death may also occur. [6] e
Therefore, it is necessary to target PMA directly to liver cells, where its activity is most required, using
some nanometric molecular drug delivery systems.
Numerous novel carriers have been proposed for reducing toxic effects of anti-malarial drugs. However,
low drug encapsulation, inadequate shelf life, and plasma instability limit their use. Among the various
carriers used for targeting of drugs to various body tissues, the cellular carriers meet several criteria
desirable in clinical applications, most importantly the biocompatibility of carrier and its degradation
product. [7] Leucocytes, platelets, neutrophils, resealed erythrocytes, and nanoerythrosomes (nEs) have
been potentially used. Among these, nEs possess great potential in drug delivery. [8] The rationale behind
the use of nEs as drug carriers is based on the certainty that senescent (damaged) cells will be removed
from circulation by phagocytic reticuloendothelial cells in liver, where targeting of anti-malarial drug is
required. The present investigation was aimed at developing and exploring the use of nEs for targeted
delivery of anti-malarial drug, PMA.
Materials and Methods
Materials
PMA was obtained as benevolent gift from IPCA Laboratories Ltd (Mumbai, India). Gluteradehyde was
purchased from SpectroChem (Mumbai, India). All other chemical and reagents used were of analytical
grade.
Preparation of erythrocyte ghost
The hypotonic osmotic lysis method described by Deloach was used for the preparation of ghost
suspension. [9] The method involves washing with a series of hypotonic saline solution, each wash being
followed by centrifugal concentration of cells. One volume of red blood cells was incubated with an equal
volume of hypotonic saline solution (0.7%). The cells were lysed and washed several times with this
solution. After each wash, the content was centrifuged at 1000 rpm for 10 min at 4±1ºC in a refrigerated
centrifuge (model Z206, Hermle, Germany) and supernatant was aspirated and discarded. The ghost
suspension was finally obtained when supernatant became colorless. The ghost cells was diluted with
0.9% saline to obtain 50% hematocrit and stored at 4±1ºC until use. [10]
Preparation of nanoerythrosomes and drug loading
An extrusion technique reported earlier [11],[12] was used with little modification for conjugation of drugs
with nEs. The erythrocytes ghost suspension (50% hematocrit) was extruded through the polycarbonate
membrane filter (0.4 μm pore, Nucleopore Corp., USA) attached to an adapter. nEs were obtained by 8-
10 consecutive extrusions and final preparation was stored at 4±1ºC in the refrigerator. PMA was
conjugated to the nE membrane using gluteraldehyde as a cross linker. Two thousand five hundred
microgram of drug was added to 2 ml of nEs preparation (50% hematocrit) in the presence of 0.3%
gluteradehyde (in a 0.9% saline solution). The mixture was incubated at 4±1ºC for 6 h and then reaction
was stopped by the addition of 2 ml of 15% glycine solution in isotonic saline. Finally, the preparation
was stored at 4±1ºC in refrigeration until used.
Optimization of formulation
The PMA-loaded nE (DnEs)' formulation was optimized on the basis of drug concentration, surface
morphology, viscosity, and sedimentation volume as follows. [13],[14],[15]
Optimization of drug concentration
To study the effect of variable drug concentration on conjugation of nEs, the various concentrations
representing 1.0, 1.5, 2.0, 2.5, 3.0 mg/ml of PMA were used. Other experimental conditions such as pore
diameter of extruder membrane (0.4 μm), gluteraldehyde concentration (0.03%), incubation period (45
min), and temperature (37±1ºC) were kept constant. Packed loaded nEs were washed with PBS (pH 7.4)
and deproteinized with acetonitrile (2.0 ml) and subjected to centrifugation at 2500 rpm (Remi, India) for
10 min. The clear supernatant was withdrawn and analyzed by HPLC using C-18 column and the mobile
phase consisted of phosphate buffer (0.05 M, pH 5):acetonitrile:concentrated perchloric acid
(750:300:2.5, v/v/v) at a flow rate of 1 ml/min. [16] The percent drug loading was determined using the
following equation:
Surface morphology
The shapes of normal erythrocytes and erythrocyte ghosts were visualized using an optical microscope
(Leica, Germany). Scanning electron microscopy (SEM Philips XL30, the Netherlands) was performed to
evaluate the surface morphology of DnEs. A small amount of DnEs were stuck on a double-sided tape
attached on a metallic sample stand, coated in argon atmosphere with a thin layer of gold, using a
POLARON E5100 SEM coating unit, and visualized for surface morphology. The vesicles size was
determined by the dynamic light scattering (DLS) method in a multimodal mode using a computerized
inspection system (Malvern Zetamaster, ZEM5002, Malvern, UK).
Viscosity and sedimentation volume
A rotatory Brookfield (Synchro Lectric LVT, USA) viscometer was used to determine the viscosity of the
formulation. Sedimentation volume of formulation was measured in a graduated measuring cylinder from
the height of sediment using the formula, F= V u/V o, where F = sedimentation volume, V u= ultimate
volume of sediment, and V o = original volume of formulation.
In vitro release rate
In vitro release studies were conducted using the dialysis membrane MW cut off 1000 Da (Sigma, USA).
Free drug from formulation was removed by centrifugation at 18000 rpm for 20 min at 4±1ºC. After
removal of free drug 1 ml of formulation was introduced into the dialysis membrane and it was placed in
a beaker containing 100 ml of 0.1 N HCl and ethanol mixtures. The beaker was kept over a magnetic
stirrer and the temperature of the assembly was maintained at 37±1ºC throughout the study. Samples
were withdrawn at defined time intervals, replenished with same volume of fresh medium, and analyzed
for drug content by HPLC. [16] Same procedure was repeated for in vitro release rate of free drug.
Stability studies
Jain and Jain (1995) [7] reported that DnEs can be stored without loss of physical integrity when
suspended in phosphate buffer saline at 4ºC for 4 weeks. Storage stability was accessed at different
temperature viz 4±1ºC, room temperature, and 37±1ºC. Formulation (DnEs) was kept at variable
temperature and changes in sedimentation volume and relative turbidity were noted periodically.
Centrifugal stress
The effect of centrifugation on sedimentation volume and drug leakage was studied by centrifuging the
formulation in centrifuge tubes at different rpm for 15 min at 4±1ºC. Drug leakage in the supernatant
solution was estimated by HPLC. [16]
Turbulence shock
It is the measure of simulating destruction of loaded nEs during injection. The formulation was passed
through a 23 gauge hypodermic needle at a flow rate of 10 ml/min, which is comparable to the flow rate
of blood. The number of passes was varied as a function of turbulence. The drug leakage in supernatant
was estimated by HPLC after fixed number of passes.
In vivo evaluation
The formulated products with promising in vitro performance were evaluated for their in vivo
performance on albino rates. All the animal studies were conducted in accordance with the protocol
approved by the Institutional Animal Ethical Committee of Dr. H.S. Gour University, Sagar. The albino
rats of either sex (average weight 250±25 g) were divided into three groups each comprising six rats.
The first group was administered drug solution equivalent to 250 μg of PMA (calculated at drug dose
level 1 mg/kg). The second group was administered with DnEs containing equivalent an amount of PMA
through caudal vein. The third group served as control. The blood samples were collected at different
time intervals from retro-orbital plexus using heparinized syringe and estimated for drug concentration
by HPLC.
Tissue distribution studies of PMA-loaded nEs were also performed using albino rats of either sex
(250±25 g). The rats were divided into three groups each comprising six rats. The animals of first group
were given drug solution (equivalent to 250 μg) of PMA through caudal vein. The animals of second
group were administered DnEs (equivalent to 250 μg) through caudal vein. The third group served as
control. After 3 and 24 h, rats of each group were sacrificed. The body organs (liver, lung and kidney)
were removed, washed to get rid of adhering debris and dried with tissue paper. The organs were
homogenized and drug concentration was determined by HPLC.
Statistical analysis
Data are expressed as the mean standard deviation (SD) of the mean and statistical analysis was carried
out employing analysis of variance (ANOVA).
Results and Discussions
The present investigation was aimed at developing a new family of natural nanoparticulate nEs for
delivery of antimalarial drug, PMA. The formulation was prepared by the extrusion method and drug was
conjugated to nEs with the help of gluteraldehyde, which served as a cross-linking agent. Glycine was
added to stop the reaction of gluteraldehyde. The formulation was optimized for effective drug loading at
variable drug concentration. The maximum percentage drug loading (25.20±1.3) was obtained with 2.5
mg/ml drug solution. Percentage loading was found to be nearly same using a 3.0 mg/ml drug solution
indicating saturation of conjugation sites [Table 1].
Table 1 :Effect of drug concentration on drug loading
Click here to view
Surface morphological studies displayed a spherical shape with a smooth surface and no aggregation
was observed [Figure 1]. No difference was observed in the morphological properties of drug-
encapsulated erythrocytes (DnEs). The size of the DnEs was found to be 125±24 nm. The viscosity of
DnEs (29.3±1.4) was almost similar to that of nEs (29.2±1.2). Sedimentation volume of unity in each
case revealed very good uniformity of the formulation. The in vitro release from the dialysis membrane
was found to be 10.02±0.8 and 53.2±1.1% after 8 h for DnEs and free drug, respectively. It was found
that the 98.51±2.4% of the free drug was released from the dialysis membrane in 20 h while only
25.57±0.9% drug was released in the case of DnEs in 20 h [Figure 2]. The decreased drug release rate
can be ascribed to cross-linking of drug to nEs by gluteraldehyde.
Figure 1 :(a) Photomicrograph of normal erythrocytes, (b) photomicrograph
of erythrocytes ghosts, and (c) SEM of drug-loaded nEs
Click here to view
Stability of formulation was assessed at 4±1ºC, at room temperature, and at 37±1ºC over time.
Formulations exhibited relatively little change in turbidity at 4±1ºC but more at room temperature and
37±1ºC indicating that the formulation is most stable at 4±1ºC. The effect of temperature on
sedimentation volume with aging was found to be negligible [Table 2]. Sedimentation volume of
formulation after centrifugation at 2500, 5000, and 7500 rpm for 15 min remained unity. This indicated
stability of formulation upon centrifugal stress.
Table 2 :Effect of aging on turbidity and sedimentation volume of DnEs
Click here to view

Figure 2 :Cumulative percentage PMA released from the free drug solution
and DnEs DnEs= PMA-loaded nEs
Click here to view
The formulation was stable against centrifugal stress as only 2.72% of drug was released after
centrifugation at 7500 rpm for 15 min from DnEs [Table 3]. The DnEs formulation was assessed for
stability during injection by subjecting to turbulence shock and was also found to withstand turbulence
shock as only 0.882% of drug was found to be released after 15 passes through 23 gauge needle [Table
3]. These studies suggested that DnEs are quite stable in nature.
The formulated product with promising in vitro performance was evaluated for in vivo performance on
albino rats. The blood serum concentration of drug solution and formulation after IV administration has
been shown in [Figure 3]. After 24 h 7.03±1.1 μg/ml of PMA was found in blood using a drug solution,
while it was found to be 5.02±0.5 μg/ml for DnEs. It shows that developed formulation has capacity for
controlled drug delivery. The tissue distribution data of PMA [Figure 4] suggested that rapid
accumulation of drug occurred in target organ such as liver (17.34±1.3 μg/ml) in 3 h in the case of DnEs,
which was almost 40% higher than free drug (12.82±0.7 μg/ml). A higher amount of drug i.e. 13.14±0.9
μg/ml was still found in liver in the case of DnEs as compared to free drug concentrations of 9.72±0.5
μg/ml, respectively after 24 h [Figure 4]. Talwar and Jain [13] also reported targeted delivery of anti-
malarial drug (primaquine) by erythrocytes and found similar higher hepatic localization of drug by
erythrocytic carriers. The higher localization of drug in target organ in the case of DnEs might be due to
removal of the carrier system by Kupffer cells of liver. Drug distribution studies in kidneys suggest higher
accumulation of drug (7.83±1.1 and 15.63±1.0 μg/ml) in kidney at 3 and 24 h, respectively in the case
of free drug as compared to 3.72±0.5 and 2.81±0.5 μg/ml in the case of DnEs [Figure 4]. The carrier
system has also retarded the excretion of PMA from kidneys.
Table 3 :Effect of centrifugal force and turbulence shock on stability of DnEs
Click here to view
Figure 3 :The blood serum concentration of the free drug solution and DnEs
DnEs= PMA-loaded nEs
Click here to view
Figure 4 :Tissue distribution of PMA at various time intervals (n=3)
Click here to view
Conclusion
Formulation, characterization, in vitro release profile, and in vivo targeting ability of the PMA-loaded nEs
formulation were evaluated in the present study. The investigations revealed an enhanced controlled
release in vitro and in vivo, higher accumulation of the drug in the case of DnEs as compared to free
drug in the target organs. In conclusion, PMA-loaded nEs as a new carrier hold promise for targeting and
systemic controlled release.
References
1.
World Malaria Report. WHO Expert Committee on malaria; Geneva, Switzerland; 2008.
2.
Farooq U, Mahajan RC. Drug resistance in malaria. J Vector Borne Dis 2004;41:45-53.
3. Molineaux L. Malaria and mortality: Some epidemiological considerations. Ann Trop Med Parasitol
1997;91:811-25.
4. Murphy SC, Breman JG. Gaps in the childhood malaria burden in Africa: Cerebral malaria,
neurological sequelae, anemia, respiratory distress, hypoglycemia, and complications of pregnancy.
Am J Trop Med Hyg 2001;64:57-67.
5. Rollo I. Resistance of Plasmodium falciparum to pyrimethamine. Transac Royal Society Trop Med
Hyg 1955; 49: 94-95.

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6. Fullerton DS. Antimalarials. 8 ed. Wilson and Gisvold's Text book of Organic Medical and
Pharmaceutical Chemistry. In: Doergae RF, editor. Lippincott, Philadelphia. 1982. p. 205-6.
7. Jain S, Jain NK. Engineered erythrocytes as a drug delivery system. Indian J Pharm Sci 1997;59:275-
81.
8. Gaudreault RC, Gicquaud C, Poyet P. Nanoerythrosome as bioactive agent carrier. US patent

5653999. Published date 5 August 1997.
9. DeLoach JR, Barton C. Circulating carrier erythrocytes: Slow-release vehicle for an antileukemic
drug, cytosine arabinoside. Am J Vet Res 1982;43:2210-2.
10. Jain S, Jain SK, Dixit VK. Erythrocytes based delivery of isoniazid: Preparation and in vitro
characterization. Indian Drugs 1995;32:471-6.
11. Jain S, Jain NK. Resealed erythrocytes as drug carriers. In: Jain NK, editor. Controlled and Novel Drug
Delivery. New Delhi: CBS Publishers and Distributors; 2002. p. 256-7.
12. Lejeune A, Moorjani M, Gicquaud C, Lacroix J, Poyet P, Gaudreault R. Nanoerythrosomes, a new

derivative of erythrocyte ghost: Preparation and antineoplastic potential as drug carrier for
daunorubicin. Anticancer Res 1994;14:915-9.
13. Talwar N, Jain NK. Erythrocytes as carriers of primaquin preparation: Characterization and
evaluation. J Control Rel 1992;20:133-42.
14. Talwar N, Jain NK. Erythrocytes as carriers of metronidazole: In-Vitro characterization. Drug Dev Ind
Pharm 1992;18:1799-812.
15. Lejeune A, Poyet P, Gaudreault RC, Gicquaud C. Nanoerythrosomes, a new derivative of erythrocyte
ghost: III. Is phagocytosis involved in the mechanism of action? Anticancer Res 1997;17:3599-
603.
16. Minzi OM, Massele AY, Gustafsson LL, Ericsson O. Simple and cost-effective liquid chromatographic
method for determination of pyrimethamine in whole blood samples dried on filter paper. J
Chromatogr B Analyt Technol Biomed Life Sci 2005;814:179-83.

Correspondence Address:
Narendra Kumar Jain
Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar
(MP) - 470 003
India
DOI: 10.4103/0973-8398.68462
Figures
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
Tables
[Table 1], [Table 2], [Table 3]

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Table 1 :Effect of drug concentration on drug loading
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Figure 1 :(a) Photomicrograph of normal erythrocytes, (b) photomicrograph of erythrocytes ghosts, and (c) SEM of drug-loaded nEs
RESEALED ERYTHROCYTES AS DRUG CARRIERS
About Authors: Varun Raj Vemula*1, Swathi Thakkalapally2, Chaitanya Kumari Bairi3
1
DepartmentofPharmaceutical Chemistry, Vikas College of Pharmacy, Jangaon, Warangal. Affiliated to Kakatiya University.
2
Department of Pharmaceutics, Care College of Pharmacy, Warangal. Affiliated to Kakatiya University.
3
Department of Pharmaceutics, TallaPadmavathi College of pharmacy, Warangal. Affiliated to Kakatiya University.
Abstract:
Carrier erythrocytes have been evaluated in thousands of drug administration in humans proving safety and efficacy of the
treatments. Carrier erythrocytes, resealed erythrocytes loaded by a drug or other therapeutic agents, have been exploited
extensively in recent years for both temporally and spatially controlled delivery of a wide variety of drugs and other bioactive
agents owing to their remarkable degree of biocompatibility, biodegradability and a series of other potential advantages.
Biopharmaceuticals, therapeutically significant peptides and proteins, nucleic acid-based biologicals, antigens and vaccines,
are among the recently focused pharmaceuticals for being delivered using carrier erythrocytes. In this review article, the
potential applications of erythrocytes in drug delivery have been reviewed with a particular stress on the studies and
laboratory experiences on successful erythrocyte loading and characterization of the different classes of biopharmaceuticals.
INTRODUCTION:
Erythrocytes, the most abundant cells in the human body, have potential carrier capabilities for the delivery of drugs.
Erythrocytes are biocompatible, biodegradable, possess very long circulation half lives and can be loaded with a variety of
chemically and biologically active compounds using various chemical and physical methods. Application of erythrocytes as
promising slow drug release or site-targeted delivery systems for a variety of bioactive agents from different fields of therapy
has gained a remarkable degree of interest in recent years. Biopharmaceuticals are among the most widely exploited
candidates for being delivered to the host body using these cellular carriers. In this review, the potential applications of
erythrocytes in drug delivery have been highlighted. [1-3]
ISOLATION OF ERYTHROCYTES:
Various types of mammalian erythrocytes have been used for drug delivery, including erythrocytes of mice, cattle, pigs, dogs,
sheep, goats, monkeys, chicken, rats, and rabbits. To isolate erythrocytes, blood is collected in heparinized tubes by
venipuncture. Fresh whole blood is typically used for loading purposes because the encapsulation efficiency of the erythrocytes
isolated from fresh blood is higher than that of the aged blood.[4] To isolate erythrocytes, blood is collected in heparinized
tubes by venipuncture.Fresh whole blood is typically used for loading purposes because the encapsulation efficiency of the
erythrocytes isolated from fresh blood is higher than that of the aged blood. Fresh whole blood is the blood that is collected
and immediately chilled to 40c and stored for less than two days. The erythrocytes are then harvested and washed by
centrifugation. The washed cells are suspended in buffer solutions at various hematocrit values as desired and are often stored
in acid–citrate–dextrose buffer at 40 c as long as 48 h before use. Jain and Vyas have described a well-established protocol for
the isolation of erythrocytes. The loading of drugs in erythrocytes was reported separately by Ihler et al. and Zimmermann. In
1979, the term carrier erythrocytes were coined to describe drug-loaded erythrocytes. [5] [6]
Advantages and disadvantages of erythrocytes in drug delivery
Advantages
Some of the most important advantages encouraging the use of erythrocytes in drug delivery include: [6-15]
1. A remarkable degree of biocompatibility, particularly when the autologous cells are used for drug loading.
2. Complete biodegradability and the lack of toxic product(s) resulting from the carrier biodegradation.
3. Avoidance of any undesired immune responses against the encapsulated drug.
4. Considerable protection of the organism against the toxic effects of the encapsulated drug, e.g. antineoplasms.
5. Remarkably longer life-span of the carrier erythrocytes in circulation in comparison to the synthetic carriers. In the
optimum condition of the loading procedure, the life-span of the resulting carrier cells may be comparable to that of the
normal erythrocytes.
6. An easily controllable life-span within a wide range from minutes to months.
7. Desirable size range and the considerably uniform size and shape.
8. Protection of the loaded compound from inactivation by the endogenous factors.
9. Possibility of targeted drug delivery to the RES organs.
10. Relatively inert intracellular environment.
11. Availability of knowledge, techniques, and facilities for handling, transfusion, and working with erythrocytes.
12. Possibility of ideal zero-order kinetics of drug release.
13. Wide variety of compounds with the capability of being entrapped within the erythrocytes.
14. Possibility of loading a relatively high amount of drug in a small volume of erythrocytes, which, in turn, assures the dose
sufficiency in clinical as well as animal studies using a limited volume of erythrocyte samples.
15. Modification of the pharmacokinetic and pharmacodynamic parameters of the drug.
16. Remarkable decrease in concentration fluctuations in steady state in comparison to the conventional methods of drug
administration, which is a common advantage for most of the novel drug delivery systems.
17. Considerable increase in drug dosing intervals with drug concentration in the safe and effective level for a relatively long
time.
18. Possibility of decreasing drug side effects.
Drawbacks:
The use of erythrocytes as carrier systems also presents some disadvantages, which can be summarized as follows: [16-20]
1. The major problem encountered in the use of biodegradable materials or natural cells as drug carriers is that they are
removed in vivo by the RES as result of modification that occurred during loading procedure in cells. This, although expands
the capability to drug targeting to RES, seriously limits their life-span as long-circulating drug carriers in circulation and, in
some cases, may pose toxicological problems.
2. The rapid leakage of certain encapsulated substances from the loaded erythrocytes.
3. Several molecules may alter the physiology of the erythrocyte.
4. Given that they are carriers of biological origin, encapsulated erythrocytes may present some inherent variations in their
loading and characteristics compared to other carrier systems.
5. The storage of the loaded erythrocytes is a further problem provided that there are viable cells and need to survive in
circulation for a long time upon re-entry to the host body. Conditioning carrier cells in isotonic buffers containing all essential
nutrients, as well as in low temperatures, the addition of nucleosides or chelators, lyophilization with glycerol or gel
immobilization have all been exploited to overcome this problem.
6. Possible contamination due to the origin of the blood, the equipment used and the loading environment. Rigorous controls
are required accordingly for the collection and handling of the erythrocytes.
METHODS OF DRUG LOADING:
Several methods can be used to load drugs or other bioactive compounds in erythrocytes, including physical (e.g., electrical
pulse method) osmosis-based systems, and chemical methods (e.g., chemical perturbation of the erythrocytes membrane).the
following are types of drug loading: Hypotonic hemolysis, hypotonic dilution, hypotonic preswelling, isotonic osmotic lysis,
Chemical perturbation of the membrane. Electro-insertion or electron capsulation, Entrapment by endocytosis, loading by
electric cell fusion, loading by lipid fusion. [21]
OSMOTIC SHOCK:
For 0.5 study, erythrocyte suspension (1 ml, 10%) was diluted & centrifuge at 3000 rpm for 15 minute. The supernatant was
estimated for % Hb release spectrophotometrically.
TURBULENCE SHOCK:
It is the measure of simulating distribution of loaded cells during injection. In this drug loaded cells are passed through a 23
gauge hypodermic at a flow rate of 10 ml/min which is comparable to the flow rate of blood. It is followed by collecting of an
aliquot and centrifugation sample is estimated. Drug loaded erythrocytes appears to be less resistant to turbulence, probably
indicating destruction of cells upon shaking.
ERYTHROCYTE SEDIMENTATION RATE (ESR):
It is an estimate of the suspension stability of RBC in plasma and is related to the number and size of the red cells and to
relative concentration of plasma protein, especially fibrinogen and α,β globulins. This test is performed by determining the rate
of sedimentation of blood cells in a standard tube. Normal blood ESR is 0 to 15 mm/hr. higher rate is indication of active
but obscure disease processes.
Use of red cell loader:
Magnani et al. developed a novel method for entrapment of non diffusible drugs into erythrocytes. They developed a piece of
equipment called a “red cell loader”. With as little as 50 mL of a blood sample, different biologically active compounds were
entrapped into erythrocytes within a period of 2 h at room temperature under blood banking conditions. The process is based
on two sequential hypotonic dilutions of washed erythrocytes followed by concentration with a hemofilter and an isotonic
resealing of the cells. There was 30% drug loading with 35–50% cell recovery. The processed erythrocytes had normal
survival in vivo. The same cells could be used for targeting by improving their recognition by tissue macrophages. [22]
Hypotonic dilution:
Hypotonic dilution was the first method investigated for the encapsulation of chemicals into erythrocytes and is the simplest
and fastest. In this method, a volume of packed erythrocytes is diluted with 2–20 volumes of aqueous solution of a drug. The
solution tonicity is then restored by adding a hypertonic buffer. The resultant mixture is then centrifuged, the supernatant is
discarded, and the pellet is washed with isotonic buffer solution. [23] The major drawbacks of this method include low
entrapment efficiency and a considerable loss of hemoglobin and other cell components. This reduces the circulation half life of
the loaded cells. These cells are readily phagocytosed by RES macrophages and hence can be used for targeting RES organs.
Hypotonic dilution is used for loading enzymes such as galactosidase and glucosidase, asparginase and arginase, as well as
bronchodilators such as salbutamol. [24] [25-26]
Chemical perturbation of the membrane:
This method is based on the increase in membrane permeability of erythrocytes when the cells are exposed to certain
chemicals. In 1973, Deuticke et al. showed that the permeability of erythrocytic membrane increases upon exposure to
polyene antibiotic such as amphotericin B. In 1980, this method was used successfully by Kitao and Hattori to entrap the
antineoplastic drug daunomycin in human and mouse erythrocytes. Lin et al. used halothane for the same purpose. However,
these methods induce irreversible destructive changes in the cell membrane and hence are not very popular. [27-29]
Entrapment by endocytosis:
This methodwas reported by Schrier et al. in 1975. Endocytosis involves the additionof one volume of washed packed
erythrocytesto nine volumes of buffer containing2.5 mM ATP, 2.5 mM MgCl2, and1mM CaCl2, followed by incubation for2 min
at room temperature. The porescreated by this method are resealed byusing 154 mM of NaCl and incubationat 370cfor 2 min.
The entrapment ofmaterial occurs by endocytosis. The vesiclemembrane separates endocytosed materialfrom cytoplasm thus
protecting itfrom the erythrocytes and vice-versa. Thevarious candidates entrapped by thismethod include primaquine and
related8–amino–quinolines, vinblastine, chlorpromazineand related phenothiazines,hydrocortisone, propranolol and tetracaine.
[30-32]
Loading by electric cell fusion:
This method involves the initial loading of drug molecules into erythrocyte ghosts followed by adhesion of these cells to target
cells. The fusion is accentuated by the application of an electric pulse, which causes the release of an entrapped molecule. An
example of this method is loading a cell-specific monoclonal antibody into an erythrocyte ghost. An antibody against a specific
surface protein of target cells can be chemically cross-linked to drug-loaded cells that would direct these cells to desired cells.
[33-34]
Loading by lipid fusion:
Lipid vesicles containing a drug can be directly fused to human erythrocytes, which lead to an exchange with a lipid-entrapped
drug. [35] This technique was used for entrapping inositol mono phosphate to improve the oxygen carrying capacity of cells.
However, the entrapment efficiency of this method is very low (1%).
RELEASE CHARACTERISTICS OF LOADED DRUGS:
There are mainly three ways for a drug to efflux out from the erythrocyte carriers: phagocytosis, diffusion through the
membrane of the cells and using a specific transport system. RBCs are normally removed from circulation by the process of
phagocytosis. The degree of cross linking determines whether liver or spleen will preferentially remove the cells. Carrier
erythrocytes following heat treatment or antibody cross-linking are quickly removed from the circulation by phagocytic cells
located mainly in liver and spleen. The rate of diffusion depends upon the rate at which a particular molecule penetrates
through a lipid by layer. It is greatest for a molecule with high lipid solubility.
DELIVERY STRATEGIES
As mentioned earlier, there are two major strategies in the delivery of drugs using erythrocytes as carriers which include
intravenous slow drug release strategy and target gene delivery.
Intravenous slow drug release strategy:
The normal life-span of an erythrocyte in systemic circulation is about 120 days. As mentioned as an advantage, in the
optimum conditions of the loading procedure, the life-span of the resulting carrier cells may be comparable to that of the
normal erythrocytes. [36] Erythrocytes have been used as circulating intravenous slow-release carriers for the delivery of
antineoplasms, antiparasitics, antiretroviral agents, vitamins, steroids, antibiotics and cardiovascular drugs among others.
[37-42]
A series of mechanisms have been proposed for drug release in circulation from carrier erythrocytes, including passive
diffusion out of the loaded cells into circulation, specialized membrane-associated carriers, phagocytosis of the carrier cells by
the macrophages of RES and, then, depletion of the drug into circulation, accumulation of the drug in RES upon lysis of the
carrier and slow release from this system into circulation, accumulation of the carrier erythrocytes in lymphatic nodes
following subcutaneous injection of the cells and drug release upon hemolysis in this sites, and, finally, hemolysis in the
injection sites. [43]
Targeted drug delivery:
RES or non-RES ‘targeting’ is another important strategy using erythrocytes as carriers.
RES targeting:
It is a well-known fact that, in physiologic conditions, as a result of the gradual inactivation of the metabolic pathways of the
erythrocyte by aging, the cell membrane loses its natural integrity, flexibility and chemical composition. These changes, in
turn, finally result in the destruction of these cells upon passage through the spleen. The other effective site for the
destruction of the aged or abnormal erythrocytes is the macrophages of the RES including peritoneal macrophages, hepatic
Kupffer cells and alveolar macrophages of the lung, peripheral blood monocytes, and vascular endothelial cells. We know that
aging and a series of other factors (e.g., stress during non-gentle loading methods) make the erythrocytes recognizable by the
phagocyting macrophages via changing the chemical composition of the erythrocyte membrane, i.e., the phospholipids
component. Therefore, a considerable fraction of carrier erythrocytes that have undergone some degrees of structural changes
during the loading procedure will be trapped by the RES organs, mainly the liver and spleen, within a short time period after
re-injection. [44-45]
A series of approaches have been evaluated to improve RES targeting using carrier erythrocytes. In one of these approaches,
the drug-loaded erythrocytes have been exposed to membrane stabilizing agents. This may increase the targeting index of the
erythrocytes to RES via decreasing the deformability of these cells. [46] [47]
Non-RES targeting:
Recently, carrier erythrocytes have been used to target organs outside the RES. The various approaches include:
* Co-encapsulation of paramagnetic particles or photosensitive agents in erythrocytes along with the drug to be targeted;
* Application of ultrasound waves;
* Site-specific antibody attachment to erythrocyte membrane.
Chiarantini et al. have reported in vitro targeting of erythrocytes to cytotoxic T-cells by coupling them to Thy-1.2 monoclonal
antibody. [48] Price et al. reported the delivery of colloidal particles and erythrocytes to tissue through micro vessel ruptures
created by targeted micro bubble destruction with ultrasound. In another study, the differential response of photosensitized
young and old erythrocytes to photodynamic activation has been studied by Rollan. [49-50]
APPLICATIONS OF RESEALED ERYTHROCYTES
Resealed erythrocytes have several possible applications in various fields of human and veterinary medicine. Such cells could
be used as circulating carriers to disseminate a drug within a prolonged period of time in circulation or in target-specific
organs, including the liver, spleen, and lymph nodes. A majority of the drug delivery studies using drug-loaded erythrocytes
are in the preclinical phase. In a few clinical studies, successful results were obtained. [51-53]
Slow drug release:
Erythrocytes have been used as circulating depots for the sustained delivery of antineoplastics, antiparasitics, veterinary
antiamoebics, vitamins, steroids, antibiotics and cardiovascular drugs.
The various mechanisms proposed for drug release include [54]
* Passive diffusion
* Specialized membrane associated carrier transport
* Phagocytosis of resealed cells by macrophages of RES, subsequent accumulation of drug into the macrophage interior,
followed by slow release.
* Accumulation of erythrocytes in lymph nodes upon subcutaneous administration followed by hemolysis to release the drug.
[55-57]
Routes of administration include intravenous, which is the most common, followed by subcutaneous, intraperitoneal,
intranasal, and oral. Studies regarding the improved efficacy of various drugs given in this form in animal models have been
reported. Examples include an enhancement in anti-inflammatory effect of corticosteroids in experimentally inflamed rats,
increase in half life of isoniazid and levothyroxine. [58]
Targeting the liver:
Enzyme deficiency/replacement therapy:
Many metabolic disorders related to deficient or missing enzymes can be treated by injecting these enzymes. However, the
problems of exogenous enzyme therapy include a shorter circulation half life of enzymes, allergic reactions, and toxic
manifestations. [59]
Treatment of hepatic tumors:
Hepatic tumors are one of the most prevalent types of cancer. Antineoplastic drugs such as methotrexate, bleomycin has been
successfully delivered by erythrocytes. Agents such as daunorubicin diffuse rapidly from the cells upon loading and hence pose
a problem. This problem can be overcome by covalently linking daunorubicin to the erythrocytic membrane using
gluteraldehyde as a spacer. The resealed erythrocytes loaded with carboplatin show localization in liver. [60] [61]
Treatment of parasitic diseases:
The ability of resealed erythrocytes to selectively accumulate within RES organs make them useful tool during the delivery of
antiparasitic agents. Parasitic diseases that involve harboring parasites in the RES organs can be successfully controlled by
this method. Results were favorable in studies involving animal models for erythrocytes loaded with antimalarial,
antileishmanial and antiamoebic drugs. [62]
Removal of RES iron overload:
Desferrioxamine-loaded erythrocytes have been used to treat excess iron accumulated because of multiple transfusions to
thalassemic patients. Targeting this drug to the RES is very beneficial because the aged erythrocytes are destroyed in RES
organs, which results in an accumulation of iron in these organs. [63]
Removal of toxic agents:
Cannon et al. reported inhibition of cyanide intoxication with murine carrier erythrocytes containing bovine rhodanase and
sodium thiosulfate. Antagonization of organophosphorus intoxication by resealed erythrocytes containing a recombinant
phosphodiestrase also has been reported. [64] [65]
Delivery of antiviral agents:
Several reports have been cited in the literature about antiviral agents entrapped in resealed erythrocytes for effective
delivery and targeting. Because most antiviral drugs are nucleotides or nucleoside analogs, their entrapment and exit through
the membrane needs careful consideration. Nucleosides are rapidly transported across the membrane whereas nucleotides are
not and thus exhibiting prolonged release profiles. The release of nucleotides requires conversion of these moieties to purine
or pyrimidine bases. Resealed erythrocytes have been used to deliver deoxycytidine derivatives, recombinant herpes simplex
virus type 1 (HSV-1) glycoprotein B, azidothymidine derivatives, azathioprene, acyclovir, and fludarabine phosphate. [66-68]
Enzyme therapy:
Enzymes are widely used in clinical practice as replacement therapies to treat diseases associated with their deficiency (e.g.,
Gaucher’s disease, galactosuria), degradation of toxic compounds secondary to some kind of poisoning (cyanide,
organophosphorus), and as drugs. The problems involved in the direct injection of enzymes into the body have been cited.
One method to overcome these problems is the use of enzyme-loaded erythrocytes. These cells then release enzymes into
circulation upon hemolysis act as a “circulating bioreactors” in which substrates enter into the cell, interact with enzymes, and
generate products or accumulate enzymes in RES upon hemolysis for future catalysis. [69] [70]
The most important application of resealed erythrocytes in enzyme therapy is that of asparginase loading for the treatment of
pediatric neoplasm. This enzyme degrades aspargine, an amino acid vital for cells. This treatment prevents remission of
pediatric acute lymphocytic leukemia. There are reports of improved intensity and duration of action in animal models as well
as humans. Other enzymes used for loading resealed erythrocytes include urease, galactose-1-phosphate uridyl transferase,
uricase, and acetaldehyde dehydrogenase. [71]
Improvement in oxygen delivery to tissues:
Hemoglobin is the protein responsible for the oxygen-carrying capacity of erythrocytes. Under normal conditions, 95% of
hemoglobin is saturated with oxygen in the lungs, whereas under physiologic conditions in peripheral blood stream only 25%
of oxygenated hemoglobin becomes deoxygenated. Thus, the major fraction of oxygen bound to hemoglobin is recirculated
with venous blood to the lungs. The use of this bound fraction has been suggested for the treatment of oxygen deficiency. 2,
3-Diphosphoglycerate (2, 3-DPG) is a natural effector of hemoglobin. The binding affinity of hemoglobin for oxygen changes
reversibly with changes in intracellular concentration of 2, 3-DPG. This compensates for changes in the oxygen pressure
outside of the body, as the affinity of 2, 3-DPG to oxygen is much higher than that of hemoglobin. [72]
Microinjection of macromolecules:
Biological functions of macromolecules such as DNA, RNA, and proteins are exploited for various cell biological applications.
Hence, various methods are used to entrap these macromolecules into cultured cells (e.g., microinjection). A relatively simple
structure and a lack of complex cellular components (e.g., nucleus) in erythrocytes make them good candidates for the
entrapment of macromolecules. [73] In microinjection, erythrocytes are used as microsyringes for injection to the host cells.
The microinjection process involves culturing host eukaryotic cells in vitro. The cells are coated with fusogenic agent and then
suspended with erythrocytes loaded with the compound of interest in an isotonic medium. Sendai virus (hemagglutinating
virus of Japan, HVJ) or its glycoproteins or polyethylene glycol have been used as fusogenic agents. The fusogen causes fusion
of co-suspended erythrocytes and eukaryotic cells. Thus, the contents of resealed erythrocytes and the compound of interest
are transferred to host cell. This procedure has been used to microinject DNA fragments, proteins, nucleic acids to various
eukaryotic cells. [74]
Advantages of this method include quantitative injection of materials into cells, simultaneous introduction of several materials
into a large number of cells, minimal damage to the cell, avoidance of degradation effects of lysosomal enzymes, and
simplicity of the technique. Disadvantages include a need for a larger size of fused cells, thus making them amenable to RES
clearance, adverse effects of fusogens, and unpredictable effects on cell resulting from the co- introduction of various
components. Hence, this method is limited to mainly cell biological applications rather than drug delivery. [75] [76]
Other applications of resealed erythrocytes include
* surface modification with antibodies
* surface modification with gluteraldehyde
* surface modification with carbohydrates such as sialic acid
* entrapment of paramagnetic particles along with the drug
* entrapment of photosensitive material
* antibody attachment to erythrocyte membrane to get specificity of action
NOVEL APPROACHES:
Erythrosomes:These are specially engineered vesicular systems that are chemically cross-linked to human erythrocytes’
support upon which a lipid bilayer is coated. This process is achieved by modifying a reverse-phase evaporation technique.
These vesicles have been proposed as useful encapsulation systems for macromolecular drugs. [77] [78]
Nanoerythrosomes:These are prepared by extrusion of erythrocyte ghosts to produce small vesicles with an average
diameter of 100 nm. Daunorubicin was covalently conjugated to nanoerythrosomes using gluteraldehyde spacer. This complex
was more active than free daunorubicin alone. [79] [80]
CONCLUSION:
During the past decade, numerous applications have been proposed for the use of resealed erythrocytes as carrier for drugs,
enzyme replacement therapy etc. The use of resealed erythrocytes looks promising for a safe and sure delivery of various
drugs for passive and active targeting. However, the concept needs further optimization to become a routine drug delivery
system. The same concept also can be extended to the delivery of biopharmaceuticals and much remains to be explored
regarding the potential of resealed erythrocytes. For the present, it is concluded that erythrocyte carriers are “golden eggs in
novel drug delivery systems” considering their tremendous potential.Most of the studies in this area are in the in vitro phase
and the ongoing projects worldwide remain to step into preclinical and, then, clinical studies to prove the capabilities of this
promising delivery system.
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Resealed Erythrocytes: As a Novel Drug Delivery Carrier

Submitted by ayushsinghal on Thu, 09/10/2009 - 20:51

a very nice prsentation,

A very nice and interesting question!!

partner-pub-6758 FORID:9 UTF-8 Search

Pharmaceutical News, Pharmaceutical Articles, and Pharmaceutical blogs for You !

Resealed Erythrocytes: As a Novel Drug Delivery Carrier

Regarding assuring the stability of products made out of resealed erythrocytes...

Thanks Mr.Jigar for the comments....

Thanks for your comments sir...

A very interesting question...

drug loading and content uniformity

• Login or register to post comments

Regarding drug loading and content uniformity:

Thanks Miss.Rujuta for the comments...

The answers to your questions are as below

a very nice prsentation, ayush!!

A very nice and interesting question!!

Answers to your questions are as below:

partner-pub-6758 FORID:9 UTF-8 Search

Skip to Main Content Area

Pharmaceutical News, Pharmaceutical Articles, and Pharmaceutical blogs for You !

Resealed Erythrocytes: As a Novel Drug Delivery Carrier

Submitted by ayushsinghal on Thu, 09/10/2009 - 20:51

Pharmaceutical News, Pharmaceutical Articles, and Pharmaceutical blogs for You !

Resealed Erythrocytes: As a Novel Drug Delivery Carrier

Submitted by ayushsinghal on Thu, 09/10/2009 - 20:51

Thanks for your comments Sir.......

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Thanks for your comments .......

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Answers to your questions:-

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Thanks for your comments Mr.Hrushikesh.......

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thanks for your comment Miss. Indira....

Clarifications a word worth while as per your questions...

Thanks for your comments Mr.Jithan.......

Regarding assuring the stability of products made out of resealed erythrocytes...

Thanks Mr.Jigar for the comments....

Thanks for your comments sir...

A very interesting question...

drug loading and content uniformity

• Login or register to post comments

Thanks Mr. Amol for your comments..

Regarding drug loading and content uniformity:

Thanks Miss.Rujuta for the comments...

The answers to your questions are as below

a very nice prsentation,

a very nice prsentation, ayush!!

Answers to your questions are as below: