Artificial Rbcs Seminar

8/9/2019 Artificial Rbcs Seminar

1/38

RASHTREEYA SIKSHANA SAMITHI TRUST

R V COLLEGE OF ENGINEERING(Autonomous Institution affiliated to VTU, Belgaum)

Department of Biote!no"o#$

A Technical Seminar Report onARTIFICIAL RED BLOOD CELLS %RBC&'() A *OTENTIAL

NANOMEDICAL DEVICE+

Submitted by

*AVITHRA NARASIMHAN%,RV-.BT-/-(

In partial fulfillment for the award of the degree

Bachelor of Engineering in Biotechnolog

Su!"ect #ode$ %&BTS'

Academic ear$ *%++*%+*

DE*ARTMENT OF BIOTECHNOLOGY

R V COLLEGE OF ENGINEERING

BA-.A/0RE12%%13

A*RIL 0-,0

RASHTREEYA SIKSHANA SAMITHI TRUST


2/38

R V COLLEGE OF ENGINEERING(Autonomous Institution affiliated to VTU, Belgaum)

Department of Biote!no"o#$

CERTIFICATE

This is to certif that the technical seminar entitled 1ARTIFICIAL RED

BLOOD CELLS %RBC&'() A *OTENTIAL NANOMEDICAL DEVICE+ has !een

presented ! *AVITHRA NARASIMHAN %1RV08BT030( at R4V4#4E, Bangalore, in

partial fulfillment of the re5uirements for the a6ard of degree of Bachelor of Engineering

in Biotechnolog during the academic ear *%++*%+*4 The contents of this 6or7, in full

or in parts, ha8e not !een su!mitted to an other Institute or Uni8ersit for the a6ard of

an degree or diploma4

Fa2"t$ In!ar#e Hea3 of t!e Department

9epartment of Biotechnolog 9epartment of BiotechnologR4V4#ollege of Engineering R4V4#ollege of Engineering

9ate$ +'thApril *%+*


3/38

ACKNO4LEDGEMENTS

I e:press m immense gratitude to Dr5 S5 Ma!e'!, ;rofessor and


4/38

TABLE OF CONTENTS

ACKNO4LEDGEMENTS888888888888888888885 i

ABSTRACT88888888888888888888888888555 iii

LIST OF TABLES8888888888888888888888885 i9

LIST OF FIGURES88888888888888888888888555 9

ABBREVIATIONS888888888888888888888888 9i

,5 INTRODUCTION8888888888888888888888,

+4+ Artificial RB#=s$ Earl attempts>>>>>>>>>>>>>>>>4444 *

05 BIOTECHNOLOGICALLY ENGINEERED H: COM*LE;ES8855 >>>>>>>>>>>>>>>44 @*4* #linical trials in patients using ;ol>>>>>>>444 &

*4 Effects of engineered >>>>>>>>44 '/5 NANOBIOTECHNOLOGICAL ENGINNERING OF H: TO

INCOR*ORATE ANTIO;IDANT EN=YMES888888888555 >

4+ ;rinciples and characteristics>>>>>>>>>>>>>>>>>>44 3

4* Animal studies>>>>>>>>>>>>>>>>>>>>>>>> ++

4 ;ol >>>>>>>>>>>4 ++


5/38

ear4 It has !een sho6n as far !ac7 as +31& that artificial RB# can !e prepared 6ith

ultrathin polmer mem!ranes of nanodimension thic7ness4 To increase the circulation

time, the firstgeneration engineered hemoglo!in (

to crosslin7

patients4 ?urther e:tension includes con"ugated

recom!inant

antio:idants to remo8e o:gen radicals to pre8ent in"ur from ischemia reperfusion4

Thus, 6e use nano!iotechnolog to prepare secondgeneration engineered

assem!ling

nanodimension solu!le comple: of polhemoglo!in (;ol

generation sstem is to prepare nanodimension complete artificial RB#s that can

circulate for sufficient length of time after infusion4 0ne approach uses lipid 8esicles to

encapsulate hemoglo!in (

pollactic acid or a copolmer of polethlene glcolpollactide (;E.;/A) to form the

mem!rane of nanodimension complete artificial RB#4

LIST OF TABLES

Ta:"e ,) #ompariti8e analsis of the four modified forms of

Ta:"e 0) #haracteristics of nanoartificial RB#Ta:"e /) #irculation time of nanoartificial RB# in human !lood

iii


6/38

LIST OF FIGURES

Fi# ,) #omposition of red !lood cellsC first artificial r!c snthesiDed

Fi# 0$(a) Artificial red !lood cell (RB#) 6ith nanodimension thic7ness nlonprotein

mem!rane4

(b)An e:ample of assem!ling of !iological molecules to form polhemoglo!in (;ol

and con"ugated hemoglo!in (

artificial cells4

Fi# /) ?our tpes of engineered hemoglo!in (


7/38

Fi#

*EGH:$ ;eglated hemoglo!in

NO$ -itric o:ide

SOD$ Supero:ide dismutase

CAT$ #atalase

LEH$ /ipid encapsulated hemoglo!in

RES$ Rectoendothelial sstemMetH:$ ethlated hemoglo!in

*LA$ ;ol lactic acid

v


8/38

vi


9/38

CHA*TER ,

INTRODUCTION

A !lood su!stitute (also called artificial !lood or !lood surrogates) is a su!stance used to

mimic and fulfill some functions of !iological !lood, usuall in the o:gencarring sense4 It

aims to pro8ide an alternati8e to !lood transfusion, 6hich is transferring !lood or !lood

!ased products from one person into another4

Fh do 6e need !lood su!stitutesG Fe can "ust loo7 !ac7 into the traged of human

immunodeficienc 8irus (


10/38

-ano!iotechnolog is the assem!ling of !iological molecules into nanodimension structures,

mem!ranes 6ith nanodimension thic7ness, or nanotu!ules 6ith nanodimension diameter4

Fi# ,$ Upper$ #omposition of red !lood cells and artificial red !lood cells4Lower left$ Red

!lood cells4 Lower right$ ?irst artificial r!c=s of + micron or larger diameterC first lipid

mem!rane nanodimension artificial r!c=sC first nanodimension !iodegrada!le polmeric

mem!rane artificial r!c=s4

,5, ARTIFICIAL RBC&' ) EARLY ATTEM*TS

The first nano!iotechnolog approach reported is the crosslin7ing of

nanodimension thic7ness mem!rane of crosslin7ed protein or proteinpolmer4 *JThis is used

to form the mem!rane of artificial RB#4 The nanodimension thic7ness mem!ranes can !e

prepared from polmers, crosslin7ed protein or crosslin7ed proteinpolmers4*JJ In this

2


11/38

form, the

same time, o:gen and car!on dio:ide can e5uili!rate rapidl across the mem!rane to

interact 6ith the enclosed materials4 *JJThe mem!ranes of the artificial RB# did not !ecome

fragile after prolonged in vitrostorage4 The artificial RB# could !e transfused 6ithout cross

matching, !ecause the do not ha8e an !lood group antigens4 Because these earl artificial

RB# are rapidl remo8ed from the circulation after intra8enous in"ection J, further research

6as carried out to sol8e this pro!lem4

Fi# 0$(a) Artificial red !lood cell (RB#) 6ith nanodimension thic7ness nlonprotein

mem!rane4 Spherical in hpotonic solution, !ecoming HcrenatedH in hpertonic solutions4

Re8ersi!le 6hen mo8ed from one solution to another4 (b)An e:ample of assem!ling of

!iological molecules to form polhemoglo!in (;ol

3


12/38

CHA*TER 0

BIOTECHNOLOGICALLY ENGINEERED H: COM*LE;ES

05, FIRST GENERATION ENGINEERED H:

The ne:t step for !lood su!stitute is to modif the a!o8e approach of crosslin7ed

glutaraldehde to crosslin7

assem!l of @1

into to:ic dimers (K+L+ and K*L*) that cause renal to:icit and other ad8erse effects4

Engineering of

recom!inant

E8en then, ;ol

significant amounts of single molecule tetrameric

#on"ugated

intercellular "unction if free from significant tetrameric

de8eloped our+3&+ !asic method of glutaraldehdecrosslin7ed

glutaraldehdecrosslin7ed human ;ol

!o8ine ;ol

4


13/38

Fi# /) ?our tpes of engineered hemoglo!in (

on nano!iotechnolog, (*) con"ugated

recom!inant

Fi#

molecules, such as intramolecularl crosslin7ed

and !ind and remo8e the nitric o:ide needed for maintaining the normal tone of

smooth muscles4 This results in the constriction of !lood 8essels4

5


14/38

Ta:"e ,) #ompariti8e analsis of the four modified forms of

050 CLINICAL TRIALS IN *ATIENTS USING *OLYH: AND *EGH:

6


15/38

.ouldHs group has de8eloped and carried out clinical trials in patients sho6ing that their

glutaraldehdecrosslin7ed human ;ol

replace e:tensi8e !lood loss43J+%JThe ha8e infused up to +% l of ;ol

trauma surger patients4 The ha8e more recentl carried out ;hase III clinical trials on &+@

prehospital emergencies patients4+%JIn this clinical trial, it is used right on the spot and in the

am!ulances as unli7e donor !lood, no tping and crossmatching is needed for ;ol

result sho6s that ;ol

;ol

e8en though there is some increase in fre5uenc of ad8erse e8ents4 +%JAs the suppl of

from outdated human donor !lood is limited, another group has de8eloped a glutaraldehde

crosslin7ed !o8ine ;ol

clinical conditions4 This includes a recent multicenter, multinational, randomiDed, single

!lind, RB#controlled ;hase III clinical trials in 2'' patients undergoing electi8e orthopedic

surger4++J6ho did not need RB# transfusion after surger 6ere 324M on the first da, &%4M up to

9a & and 134@M all the 6a to follo6up4 ++JThis !o8ine ;ol

routine clinical use in patients a num!er of ears ago in South Africa, a region 6ith higher

incidence of


16/38

05/ EFFECTS OF ENGINEERED H: ON THE HEART AND VASOACTIVITY

Vasoconstriction of !lood 8essels in the heart is the 7e 5uestion raised ! the metaanalsis4+JFe, therefore, carried out studies in rats to see if there is 8asoconstriction of

coronar 8essels as sho6n ! the ischemic electrocardiogram (E#.) changes of the heart in

the form of ST ele8ation4+@JRat hearts ha8e 8er high heart rates and therefore more sensiti8e

to ischemic changes !ecause of 8asoconstriction4 Fe prepare ;ol

containing different percentages of unpolmeriDed

glutaraldehde crosslin7ing and characteriDed to ensure that the all ha8e the same o:gen

affinit4+@J0ur analsis of the result sho6s that ;ol

molecules 6ould not cause coronar arter 8asoconstriction4 Fith increasing percentage of

single

Fith e8en higher percentage of tetrameric

arrhthmia4 ST ele8ation could !e due to 8asoconstriction resulting in a decrease in the

suppl of o:gen to the heart and this ma e:plain the o!ser8ation of small su!endocardial

lesions in some primates and s6ine after infusion 6ith one tpe of modified

+%%M single

different tpes of engineered

com!ining the clinical results of different tpes of engineered

Another group has sho6ed that inhalation of nitric o:ide could pre8ent

8asoconstriction in rats that recei8ed a tpe of engineered

in 8asoconstriction4+1JThis has important implication especiall for use in surger4 It ma

also allo6 the possi!le use of those engineered

8asoacti8it44 ?urthermore, their result also support the role of nitric o:ide in 8asoacti8it

related to some tpe of engineered

CHA*TER /

8


17/38

NANOBIOTECHNOLOGICAL ENGINEERING OF H: TO

INCOR*ORATE ANTIO;IDANT EN=YMES

/5, *RINCI*LES AND CHARACTERISTICS

?irstgeneration engineered

clinical applications4 There are other conditions 6ith sustained lac7 of o:gen suppl, as in

coronar arter diseases, sustained hemorrhagic shoc7, stro7e, mocardial infarction, organ

transplantation, and other conditions4 Engineered

ischemia situations this 6ill also result in the production of o:gen radicals that can cause

tissue in"uriesOa condition called ischemiareperfusion in"ur4+2J Fe, therefore, use

nano!iotechnolog to assem!le

form ;ol


18/38

small amount of crstalline #AT and S094+&J*%J Using the method as descri!ed, the

percentage of the original enDme acti8ities after crosslin7ing is 3133M for S09 and '1

3%M for #AT4+&J#rstalliDed enDmes are e:tremel e:pensi8e and in order to allo6 for scale

up production, 6e recentl 6ere a!le to crosslin7 hemolsate (S?

S?

S094 If needed, more #AT and S09 can !e e:tracted from the hemolsate and added to the

hemolsate to prepare PolySFHb!"S#$ that contains a higher concentration of the

enDme4*+JUnli7e ;ol

pero:ides, and sta!iliDe the crosslin7ed

heme release4+&J+'J+3J*%J*+J?or clinical applications, further de8elopments are needed for scale

up, !ut this can !e !ased on the standard scale up method used for ;ol

infecti8e agents should not !e a pro!lem !ecause methods are a8aila!le to prepare

intra8enous S09 and ;E.S09 preparations that are alread !eing used in patients4

Fi# @) Effect of supero:ide on the sta!ilit of$ (Upper);ol

hemoglo!in (

(Lower right)crosslin7ed

more enDmes e:tracted from RB#s added4

/50 ANIMAL STUDIES

?ree S09 and #AT are remo8ed rapidl from the circulation 6ith a halftime of less than %

min4 In the form of ;ol

10


19/38

compara!le to ;ol

reperfusion of ischemic rat intestine, ;ol

o:gen radicals caused ! ;ol

+3JStudies ha8e also !een carried out on glo!al cere!ral ischemia reperfusion in a

hemorrhagic shoc7 model4+'J ;ol

!lood !rain !arrier (BBB) disruption 6hen compared 6ith saline, S?

solution of free

significant !rain edema in those treated 6ith saline, S?

6ith coronar arter diseases ma re5uire ;ol

a8oid possi!le damage to the heart as o!ser8ed in some cases4 0ther conditions 6here

ischemiareperfusion in"uries 6ould !e more li7el include organ transplantation !ecause

stored donor organs are in a state of o:gen lac74 0ne group found that the use of this

;ol

studies4**JThe potential of engineering

to the use of

/5/ *OLY H: FIBRINOGEN AND *OLY H: TYROSINASE

In addition to ;ol

6ith other enDmes or proteins are also possi!le4 ?or e:ample, high !lood 8olume loss, large

8olume RB# replacement alone 6ould not replace the platelets and coagulation factors and

there is a need to replace these also4 Fe, therefore, use nano!iotechnolog to de8elop a !lood

su!stitute that is an o:gen carrier 6ith plateletli7e properties4 This is a no8el !lood

su!stitute, 6here

comple: of ;ol

6orse clotting pro!lems 6ith further increase in the !lood replaced4 In another stud, 6e use

;ol

11


20/38

;ol

the decrease in sstemic trosine le8el4 The result is a significant decrease in the rate of

gro6th of the melanoma in a rat model4*1JBoth concepts ha8e !een sho6ed in animal studies

!ut the final test 6ould re5uire scale up and de8elopment for clinical trials in human4

CHA*TER

Artificial Rbcs Seminar

Documents