Resealed Erythrocytes: As a Novel Drug Delivery Carrier

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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.

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Comments

Shreesha Bhat's picture

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

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Priyanka .p.bhosale's picture

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

Rujuta Mehendale's picture

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, anti- parasitics or even hepatic application based drugs) using this technology?

Thanks!

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Amol Malpani's picture

Dear Ayush,
resealed erythrocytes is having potential but what about drug loading and content uniformity? Suggest some remedies.
Regards,
Amol

Ayush Singhal's picture

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

NIRAJ's picture

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

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Jigard Patel's picture

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????

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Jithan Aukunuru's picture

Hi,

Good presentation. Can you assure the stability of products made out of resealed erythrocytes?

Jithan

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Lakshmi's picture

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.

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Lakshmi's picture

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..

Dr.S.Gunasakaran's picture

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. Head - Clinical Research & Medical Affairs www.clinicalresearchsociety.org/forum

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Sirisha Pingali's picture

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

Viswanadha Institute of Pharmaceutical Sciences.

www.vnips.edu.in

Ayush Singhal's picture

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 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.
-----------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 http://www.pharmainfo.net/ayushsinghal/biography

Sirisha Pingali's picture

dear ayush..
Good explanation..

Sirisha Pingali

http://www.pharmainfo.net/sirisha

Viswanadha Institute of Pharmaceutical Sciences.

www.vnips.edu.in

Hrushikesh Karandikar's picture

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

Ayush Singhal's picture

Answers to your questions....

a) 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.

b)NO we cant make resealed erythrocytes available in blood banks because of :-

---The lack of reliable and practical storage methods .

---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.

thank you....

Ayush A. Singhal RPCP, CHANGA GUJARAT http://www.pharmainfo.net/ayushsinghal/biography

Kunal Ingale's picture

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?

Ayush Singhal's picture

Answers to your questions:-

1)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.

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 http://www.pharmainfo.net/ayushsinghal/biography

Khushbu Hasmukh Patel's picture

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

Ayush Singhal's picture

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,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 http://www.pharmainfo.net/ayushsinghal/biography

Ganesh Nirma's picture

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.

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Nikhil's picture

Can you explain detail about electro encapsulation method of drug loading? is there any disadvantage?

Nikhil

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Apurva Anugamini's picture

hello,
What do you mean by heparinised tube?

Ayush Singhal's picture

--------------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 http://www.pharmainfo.net/ayushsinghal/biography

Chitrak Aladhuri's picture

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

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Ankit's picture

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

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Ankit Sheela Mittal's picture

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

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Aarti Anantram's picture

Mr. Singhal,
Do the effects of drug incorporated into erythrocytes persist for the entire lifespan of the erythrocyte?
Regards,
Aarti

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

VENKATA KIRAN KUMAR MANDLEM's picture

IS it possible to delivery the antibodies that target the cancer through resealed erythrocytes. If so what are the barriers.

Ayush Singhal's picture

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 http://www.pharmainfo.net/ayushsinghal/biography

Vijaya Sreedhar S's picture

how can u separate rbc from plasma ? how can u confirm the resealing of the drug in rbc? is the process should be carried out at aseptic area? how much rbc sample was taken for loading of drug?