Non-viral gene delivery systems

67 Pages·2017·0.89 MB·English

REVIEW
1
Department of Pharmaceutics , Faculty of Pharmacy, Alexandria University, Alexandria, Egypt; Department of
2
Pharmaceutical Sciences , Appalachian College of Pharmacy (ACP), Oakwood, VA, USA; Department of Pharmaceutical
3
Sciences , School of Pharmacy, University of Saint Joseph (USJ), Hartford, CT, USA
Non-viral gene delivery systems: hurdles for bench-to-bedside
transformation
1 1 1 2 1 3,
N. A. HelAl , A. OsAmi , A. Helmy , T. mcDONAlD , l. A. sHAAbAN , m. i. NOuNOu *
Received May 23, 2017, accepted July 28, 2017
*Corresponding author: Mohamed Ismail Nounou, MPSc., Ph.D., School of Pharmacy, University of St. Joseph, 229
Trumbull Street, Hartford, CT 06103, USA
REVIEW
suffered from a massive immune response triggered by the Suspea rokf Therapeutics announced that the medicine had succeeded
a high titer of the adenoviral vector used as the carrier systeinm af olra te-stage clinical trial in treating an inherited eye disease
gene transportation, leading to multiple organ failure . The se(invehreer ited retinal dystrophies) that can cause�blindness (Bennett
immune reaction caused his death four days after the initieatt iaol.n 2 016).
of the study S( ibbald 2001�.)The tragic eG lsinger incident was aI n spite of such continuing advancements and promising clinical
®
major setback to the gene therapy research field . The numbdear toa,f it apparently took only five years for Glybtoe rfaizzle out
peer-reviewed original research articles and clinical trials adfrtaesr -failing commercially. On April 2017, UniQure announced
tically declined in the couple of years following this incidethnacte it would no longer seek to renew marketing authorization
®
2( 000-2006 .) The field started to regain traction in 2006, afteorf alipogene tiparvovec (Glyber) aafter the Western worlds’
recovering from this incidence E( delstein et al . 2004 .) first five-year conditional approval of the therapy is set to expire
Another drawback for the viral vectors is the insertional mount aO- ctober 2017 (European Biotechnology Life Science and
genesis, disrupting the expression of tumor suppression geInndeu, sotr y Magazine 2017). UniQure is set to pull the drug from
activating an oncogene leading to the malignant transformtahteio mn arket and wind down all the related operations and infra-
of cells (Nault et al. 2015). However, three adenoviral-based structure. The reason behind this decision is, unfortunately, not
drugs have reached the market: Gend® i,c Gliynbeera ® and Strimv- related to efficacy or safety, but due to weak sales and high price
elis® . Gendicine ® is a recombinant Ad-p53 gene therapy for hetaadg . Glybera ® was a commercial failure. The decision brought a
and neck squamous cell carcinoma (HNSCC). Gendic® inweas unique odyssey for UniQure to an end. Such a decision could
approved via the State Food and Drug Administration of Cbhein a set back to the field for some while and definitely would
in October 2003 (Frew et al. 2008). The second gene therapiyn spire the gene therapy researcher and scientists into exploring
® cheaper, more efficient, and safer options. Non-viral carriers
formulation to be marketed worldwide was Gly b(aelirpaogene
® could represent a cheaper and easier to manufacture option as
tiparvovec). Glybera was approved by the European Union (EU )
compared to viral vectors with its unrealistic price tag (~ $ 1
as an engineered adeno-associated viral vector. Although Gendi-
®
® million for Glybera ) (Han and Ni 2015; Morrison 2015; Ronfard
cine was approved for sale in China in 2003 for cancer treatment,
® et al. 2017).
Glybera (alipogene tiparvovec) was finally approved in Europe in
On the other hand, non-viral gene delivery vectors are generally
2012 for an ultra-rare genetic disease, familial lipoprotein lipase
® less toxic and immunogenic compared to viral vectors oJ( nes et
deficiency, nine years after Gendici(Fneerreira et al. 2014).
® al . 2013 ; Nayerossadat et al . 2012 .) They are safer and less expen-
Glybera was developed by UniQure Inc. UniQure went through
sive . oH wever, non-viral gene delivery has low efficiency and less
major hurdles pushing alipogene tiparvovec through the clinical
ability to target gene expression to the area of pathology impeding
testing and approval process in Europe (Salmon et al. 2014; Scott
their use K( atz et al . 2013 ; aY ng 2012 .) Non-viral gene delivery
2015; Steinhagen-Thiessen et al. 2016). As mentioned eianr lier,

includes lipid-based vectors and cationic polymers . They have been
the early 2000tsh,e field of gene therapy was affected greatly by

used for the transfer of nucleic acids for many years . The cationic
the multiple toxicity and death incidents during clinical trials in
lipid DOTMA N( -1[ -2( ,3-dioleyloxyp) ropyl-] N,N,N-tri-methylam-
Europe and USA and went through its own roller coaster ride,
monium chloride ) showed high efficiency in delivering RNA in
trying to overcome past safety and other problems to prove its
various cell lines and, to a lesser extent, DNA transfection M( alone
worth as a viable therapeutic option. In the time frame between
® et al . 1989 .) Early examples of cationic polymers used for DNA
2003 and 2012, Glybera was rejected by European regulators
transfer are PEI p( olyethylenimine ) and PLL p( oly-l-lysine .)
three times (Salmon et al. 2014). In spite of the hurdles, gene
Cationic polymers undergo diverse modifications that we will
therapy was gaining momentum via technological advances
detail later in the review.
reflected in improved engineered viruses, or “vectors” that help
®
deliver the treatment along with the public need. Glywbaesra
®
finally approved in Europe in 2012. Glybe wraas initially priced 2. Requirements for a successful gene delivery
at around $1 million in Europe (Han and Ni 2015; Morrison 2015 ;
A successful delivery system depends on the vector or carrier
®
Ronfard et al. 2017). Glybera represented the initial spark in the
used in the safe transport of the genetic material into its final
field of gene therapy and has fueled the field since 2012. Another
cellular target destination. Vectors are vehicles that deliver the
gene therapy, from GlaxoSmithKline™ (originally designed and
genetic material to target cells. Selectivity and efficiency in
developed by San Raffaele Telethon Institute for Gene Therapy
delivering a gene to the target cell along with minimal toxicity
®
and the biotech MolMed in Italy), is Strimv, ewlishich was
are the most crucial criteria. The lesson history taught, based on
developed for a rare immune deficiency. It was approved in 2016
the failed and deadly clinical trials, is that safety perhaps has a
(European Commission) (Monaco and Faccio 2017; Ronfard et
leverage over efficacy (Smith and Byers 2002; Somia and Verma
al. 2017; Schimmer and Breazzano 2016; Touchot and Flume
2000; Stolberg 1999). Vectors should also be produced in large
®
2017). Strimvelis isan ex-vivo stem cell gene therapy designed
amounts with low cost. Carriers can largely vary in their trans-
to treat a rare inherited disorder (around 15 European children/
fection efficiencies and toxicity profiles. Furthermore, there is
year), severe combined immunodeficiency due to adenonsoin oe ne delivery vector that is ideal for the standards of a perfect
deaminase deficiency (ADA-SCID) (Gardner 2016; Hoggatt gene delivery. The solution lies in the modifications that can be
2016; Levenson 2016; Shorter et al. 2017). ADA-SCID is a done to obtain a smart delivering vector, possessing considerable
homozygous disease resulting from a rare genetic dissourdpeprl y for the previously mentioned principles (Boeckle and
affecting children. It results in an ill-functioning immune sysWteamg ner 2006).
and inability to fight off common infections. Aside from bAimg ong all vectors, viral vectors possess the highest transfection
®
Pharma players like GlaxoSmithKline™, Glybera inspired efficiency . oH wever, it has detrimental side effects of cytotoxicity
numerous gene therapy startups such as Spark Therapeauntdic as ,h igh ability to induce the bodys’ immune response lG( over
BioMarin Pharmaceutical, and Bluebird Bio (Carr and Bradsheat wal . 2005 .) Non-viral techniques for gene delivery are direct phys-
2016; Cucchiarini 2016; Han and Ni 2015; Kotin and Snyder ical methods such as microinjection and particle bombardment, i.e.
2017; Morrison 2015; Ronfard et al. 2017; van der Loo and gene gun, electroporation, sonoporation, laser beam and magne-
Wright 2016). Clinical data and initial controlled experimenttao-fection, and the chemical-based approaches such as non-viral
tion in humans from these startups suggest that gene thercaapryri eisr systems L( iposomes, lipoplexes, polymers, peptides,
potentially beneficial in multiple diseases, such as hemopnhailniao,p articles, etc .) Physical methods are the easiest to implement
beta thalassemia, and sickle cell disease (Cucchiarini 20a1n6d ; have acceptable transfection efficiency . nU fortunately, phys-
Ronfard et al. 2017). Furthermore, what could become the firsict al methods require numerous doses and cause damage to certain
gene therapy to win approval in the United States moved closetyr ptoes of cells M( ali 2013.)
market in 2015 with the first successful randomized, contrColhledm ical methods are also generally easy to prepare, but their
trial for any gene therapy aimed at an inherited disease, wushee nis limited due to their insufficient transfection efficiency . As
628 Pharmazie 72 2( 01)7
REVIEW
a ,tluser taerg noitneta dluohs eb detcerid ot eht esu fo dirby h enely locylg EP( ,)G ly porpyxordy h edimalyrcahtem ,)AMPH(
.smetsys gnisU a lariv rotce v gnola htiw eno fo eht citehtnys ,sra gus dna snietorp era .desu ro F ,elpmax e eca frus enely hteylop
sreirac nac ezimitpo eht s y’rotce cneici v f fe htiw rew ol -xototy c locylg gnitaocEP( yle v )noitalyG itcef sesaerced fe eht snoitcaretni
esnopser enumi eht ecuder ot desu sa w locylg enely hteyloP . ytici fo dolb stnenopmoc dna e v itadarg ed semyzne htiw eht rotce v
suriv oneda gnisu smetsys desab-lariv ni y cneici f fe eht ecnahne dna
,eca frus gniref fo degnolorp noitalucric ni eht .dolb ,eromrehtruF
W( nana gno dnaC ely or102 .)0 gnolA htiw dirby h ,smetsys trams
gniteg rat sdna gil gnidulcni ,snietorp ,seidobitna gnitartenep-lec
S . y pareht eneg fo dlro w eht otni decudortni neb e v ah sreirac tram
,seditpep ,sra gus dna rehto lams elucelom sdna gil nac eb desu ot
,sremylop hcihw nac yalpsid taerg segnahc ni rieht seitreporp ro
102 .la te senoJ( sreirab eseht emocre v o T ;3 102 .la te na .)6
eht eb nac ,tnemnoriv ne eht ni ilumits ro segnahc eltil ot eud epahs
suelcun ni eht ngised fo le v on trams eneg reirac .smetsysS tram
sreirac nac yfidom rieht epahs dna scitsiretcarahc ot dio v ya nam 3.3. Cellular level
.)502 .la te recniD( ylisae sreirab lec
htoB desab- remylop dna desab-dipil sreirac yrac ev itisop
Non- v i r a l g e n e d e v rl yi s y s t e m s w i l l b e t h e f o c u s o f o .u r v wr i e e
seg rahc ot( elbane x elpylop ro x elpopil noitamrof htiw eht -a g en
A n e v or v w i e o f t h e s y s t e m s u s e d , a l o n g w i t h t h e r e c e n t en v lo
deg rahc-yle vNDit ;)A ,erofereht noitprosda nac sya w la e kat ecalp
a a d t n v e c n c e b w o h d e i n ni l o- on l lv v, l te g vi o hd e ir g e n na i r e l e a syp
newteb eseht snoitac dna deg rahc-yle eht v lec ita g en .enarbmem
d i s c uRse d . r ge e sci p eosa pa atnm pta rdese hte r c-i ere hne ns d c t e s
y na retne ot lairetam citeneg a rof sya w o wt era ereht ,eromrehtruF
b a c k o f g e n e t h e r a yp r e s e a r c h f r o m 2 0 0 6 t o d a t e a r e s u yr e ev d ,
-odne yb ro ”sdohtem lacisy hp“ yre v iled tcerid yb rehtie ,lec teg rat
c a t g e o r i z e d , a n d t au bl a t e d i n A p p e n d i x 1 (9 0 r e s e a r c h p a p e r s )
.sisoty c R detaidem- rotpece ,sisoty codne hcihw sedulcni nirhtalc
A p a p n e d n d i x 2 ( 5 4 r g e i s t e r e d p a t e n t s ; f o r a p p e n d i c e s s e e o n l i n e
dna ealoe v ac detaidem ,sisoty codne ,sisoty conip-orcam dna
e v r s i o n ) . In t h e s e t a b l e s , r e s e a r c h p a p e r s o r p a t e n t s a r e s u m m a -
,srehto si deredisnoc eht tsom nomoc e katpu msinahcemV( tgio
r i z e d b a s e d o n t h e n o n - v i r a l c a r r i e r s y s t e m u s e d , c a r r i e r s y s t e m
te .la102 .)4T eh melborp gnidra g sisoty er codne seil ni semososyl
d e s i g n , a p p l i c a t i o n s , i f c e a f y c t e s t i n g , t o x i c i t y t e s t i n g , x e p r e s s i o n
gnihcata ot eht semosodne gnidael ot noitadarg ed fo eht citeneg
r e a d o u t t e c h n i q u e s , r e p o r t e r a n d t h e r a p e u t i c g e n e s u s e d , p h y s i -
lairetam lilahK( te .la .)602 ,oslA sisoty cogahp si elbacilpa rof
c o c h e m i c a l c h a r a c t e r i z a t i o n o f t h e c a r r i e r s y s t e m , s t u d y d e s i g n ,
a m o u n t o f g e n e t i c m a t e r i a l u s e d , a n d g o l d s t a n d a r d s a n d c o n t r o l s segahporcam dna citirdned .slecT ,erofereh lamosodne epacse fo
u s e d i n e a c h . s t u d ySuch s u m m a r y ow u l d e a s i l y a i d r e s e a r c h e r s eht sreirac ni lariv-non smetsys si deredisnoc a gib egnelahc rof
t o fe o f r t l e s s l y c o m p a r e t h e a r v i o u s d e s c r i b e d c a r r i e r sy s t e m s . eht ralulecartni yre v iled fo cielcun .sdica E lamosodn epacse fo
Thes e t a b l e s c o u l d b e o f a l v u e t o x e p e r t s a n d w n c e o m e r s i n t h e eht sreirac nac de eb v aiv eihca gnida a gnitartenep-lec ,editpep
i ef l d o f n o n - v i r a l g e n e d e v rl yi r e s e a r c h . lamosodne enarbmem erutpur hguorht erop ,noitamrof ro notorp
102 .la te gneM( tcef fe egnops .)7
3. Non-viral gene delivery: Breaking down the barriers
T eh noitalumrof fo na e v itcef fe eneg yre v iled rotce v sretnuocne 3.3.1. Endosomal escape strategies
ynam seldruh gninig eb morf noitazilanretni dna gnipacse -arg ed
1.3.3 1. . Ttcef fe egnops notorp eh
noitad tneici gnisap se .noitcefsnart f yb v dnaI ytefas fe itcepsrep n
T eh notorp egnops tcef fe si eht noitcarta fo snotorp gnitluser ni
siht trap fo , eht w eseht eiv er ,smelborp htiw detcepsus ,snoitulos
htiw gnola emosodne eht ot reta w dna edirolhc e v isecx e fo ecnartne
tneref lA eneg liw fid refsnart eb e sdohtem .desucsid v dluohs ah
eht ytiliba ot reuqnoc o wt rojam :stniartser ,tsri f eht den ot yrac eht ,snotorp ecneh gnisaercni s eht ezis ’emosodne ylacitsard dna
102 .la te niJ( sksir laminim htiw teg rat sti ot lairetam citeneg eht ;4 1 rheB( erutpur rieht ot gnidael yltneuqesbus T .)79 y hposolihp eh
N tadasoreya te .la 102 ,)2 dna ,dnoces eht ytiliba fo eht cielcun ni eht ngised fo a lufsecus lariv-non eneg reirac metsys si eht
dica ot etartenep lec .senarbmem ro F a hgih etar fo lec -cefsnart , yradnoces , yramirp sa hcus( spuorg detanotorp-ylhgih fo noisulcni
,noit eseht o wt snoitidnoc tsum eb deredisnoc gnirud noitalumrof dna yraitret gnidliu )senima skcolb otni fo eht eht b reirac .metsys
taht sreirab ralulecartx e gnimocre v o sediseb rotce v citeneg eht fo S hcu detanotorp spuorg dluo w dia eht noitaerc fo notorp egnops
edulcni citamyzne noitadarg ed dna dipar ecnaraelc retfa -nimda reirac eht htiw gnola selucelom edirolhc dna reta w gnitcarta tcef fe
noitartsi gnauH( te .la102 .)5 In-vivo� yre v iled sniamer eno fo eht -enely hteyloP .erutpur sti ot gnidael ,semosodne eht nihtiw metsys
tsom detacilpmoc stsetorp ni eneg y pareht sa eht dengised rotce v
EP( enimi notorp eht etareneg ot .la teI rheB yb dengised neb sah )
tsum epacse eht lailehtodne-oluciter metsysR( ES ) retfa cimetsys
egnops tcef fevia , , yramirp yradnoces dna yraitret ,senima dna ot
noitartsinimda sediseb gnisorc ynam rehto sreirab erofeb
yltneuqesbus etareneg hguone gniref fu b yticapac ot yltneici f fe
102 .la te senoJ( slec teg rat gnihcaer nw ohs sa ,nosaer siht ro F .)a3
tcefsnart citeneg slairetam rheB( 1 ;69 fisuoB te .la 1 .)59T eh
secidnepA ni1 dna ,2 y nam srehcraeser sapyb eht esu fo mures
gniref fu b yticapac si laitnese ni gnisaerced eht cidica Hp fo eht
ni riehtin-vitro ycaci dna f ytefas fe gnitset dna tcelg y en nain-vivo
ot dnet semososyl ,msinahcem x elfer a sA .tnemnoriv ne lamososyl
.smetsys le v on rieht fo noitatnemirepx e
esaerced s eht Hp ’emososyl ot niatniam sti cidica tnemnoriv ne ni
eneG yre v iled sreirab nac eb dei fisalc otni ruof ’sle v el ,sreirab
fo esaercni e v isecx e ot gnidael ,secorp noitaci fidica suounitnoc a
:sw olof sa
eht s ’emososyl reta w tnetnoc dna yltneuqesbus ezis gnidne ni sti
1 rheB( erutpur 1 .la te fisuoB ;69 EP .)59 I dlog a deredisnoc si
3.1. Blood level
dradnats ni lariv-non eneg yre v iled ni smret fo , y caci f fe ylniam
TN .esaelcun emyzne eht sniatnoc mures dolb eh edarg ed sesaelcu esuaceb fo sti gniref fu b yticapac dna sti notorp egnops .tcef fe
eht ralulecartx e cielcun sdica fi , .detcetorpnu re stnemele v oeroM ro F ciremylop sreirac tuohtiw Hp gniref fu b ,seitreporp hcus sa
ni eht dolb ro mures snietorp yam x elpmoc htiw eht , remylop nasotihc dna enisyl-l-ylop ,)LP( lanoitcnuf seiteiom era deda
R( desoty cogahp si taht ylbmesa na gnimrof 102 .la te da .)4
ot meht ot e v orpmi rieht gniref fu b yticapac dna ecnahne rieht
noitcefsnart .seicneici f fe eniditsiH si eno fo eht yltneuqerf desu
3.2. Tissue level seiteiom sa a Hp gniref fu b gnicnahne lanoitcnuf puorgS( ih te .la
102 ;3S nu dna uohZ102 rehtonA tnega hcihw si ylediw desu si .)6
N ci ficepsno noitcaretni fo eht remylop htiw eht ralulecartx e
.eniuqorolhcC eniuqorolh si a ciportomososyl tnega taht stne v erp
xirtam srednih eht w olf fo eht remylop gniyrac eht citeneg
lamosodne noitaci fidica dna strapmi gniref fu b fo eht lamosodne
lairetam irasoD-lA( dna oaG .)902N ci ficepsno noitcaretni si na
,Hp gnitepmoc htiw eht suounitnoc noitaci fidica secorp dna
eht fo ezis eht ot gnidroca yltneref fid detcef fa eb nac taht elcatsbo
T . rotce v .x elpylop fo tneme v om eht srednih ezis elcitrap eg ral eh yltneuqesbus gnidael ot a kaep esir ni ytiralomso dna ylautne v e
dolB dna eusit sreirab era deman eht ralulecartx e .sreirab citomso sisyl fo eht lamososyl .elcise vC eniuqorolh setalumuca
detna wnU snoitcaretni ta htob sle v el nac eb dedio v a yb emos dna semosodne sa hcus ,lec eht fo stnemtrapmoc cidica eht edisni
snoitaci fidom no eht s ’rotce v .eca frus seiteioM hcus sa -hteylop W( semososyl S dna treflo 1 ruomy e .)89
7 eizamrahP 2102( )7 926
REVIEW
33. 1. 2. . Cell penetration peptides C( PPs) easily tailor-design the carrier system to minimize and control
the system toxicity profile .
CPPs are short sequences of amino acids, mainly 10-30 residues
M( adani et al . 2011 .) CPPs are able to cross the plasma membrane
of living cells to facilitate the transportation of hydrophilic m5a.c rNo-on-viral gene delivery carriers: Methods and tech-
molecules such as proteins, peptides, and nucleic acids . Mnoisqtulye,s
CPPs are classified into two main categories . The first is the
The main core of gene delivery is to safely and effectively transfer
cationic peptides that usually contain arginine and lysine residues,
the genetic material to host target cells . There are three main
eg. ,. TAT peptide, penetratin and oligoarginines . The second is the
methods for delivering genetic material to the target cells: phys-
amphipathic peptides that consist of both hydrophobic and hydro-
ical, chemical, and biologically-responsive methods N( ayerossadat
philic segments M( adani et al . 2011.)
et al . 2012.)
33. 1. 3. . Pore formation
5.1. Physical methods (direct delivery)
Pore formation is another mechanism to facilitate the endosomal
Physical methods are means by which nucleic acids are directly and
escape of non-viral gene delivery systems . This mechanism can be
rapidly delivered via different methods . Some of these methods are
achieved via peptides such as AG LA peptide g( lutamic acid-ala-
summarized as follows:
nine-leucine-alanine ) N( ir and Nieva 2000 .) Pore-forming peptides
become incorporated into the vesicle bilayer structure and accu-
mulate to form a pore with a diameter ranging from 5 to 105 .1 .Å .1. Microinjection
AG LA can only be added as an additional functional component to
Microinjection is the injection of an intended gene, drug, or
polyplexes or lipoplexes N( ir and Nieva 2000.)
molecule into the skin using micro-needles . Purified DNA was
successfully microinjected for the first time in 1980 K( ucherlapati
3.4. Nuclear level et al . 1984 .) In addition, micro needles were used to deliver oligo-
nucleotides, insulin, proteins, and RNA . A micro-needle is a mean
Entering the nuclear envelope efficiently requires a molecule
that directly pervades the skin in the least possible intrusive way
of a small mass and diameter. Molecules with a mass of less
P( rausnitz and Langer 2008.)
than 50 kDa and a diameter of few nanometers can cross the
nuclear membrane by passive transport (Alberts et al. 2002) .
Comparing this to the size of the DNA, the particles that 5c.1a.n2 . Gene gun
pass the membrane passively are smaller than the transported
The target nucleic acid is precipitated onto gold or tungsten parti-
DNA (Alberts et al. 2002; Khalil et al. 2006), a problem that can
cles, then these particles are bombarded with a high velocity to the
be overcome by an active transport system. A nuclear localiza-
intended organ or cell using a biolistic device g( ene gun ) M( ellott
tion signal (NLS), mostly a peptide, is either coupled with the
et al . 2013 .) Particle bombardment was first known and applied
transported nucleic acid or attached on the surface of the carrier .
in 1992 when DNA-coated tungsten molecules were delivered to
NLS aids the entrance of the carrier by energy mediated nuclear
plant cells F( iner et al . 1992 .) This method was proven to have a
translocation. It is also said that macromolecules may pass the
great role in gene immunization L( oehr et al . 2000 .) oH wever, it
nuclear membrane during the cell mitosis as the nuclear envelope
must be understood that particle bombardment may cause tissue
cracks (Fortier et al. 2014). Sequence PKKKRKV in the SV40
injuries M( ellott et al . 2013.)
(Simon Virus 40) Large T-antigen, Nucleoplasmin (AVKRP-AAT
KKAGQAKKKKLD), EGL-13 (MSRRRKANPTKLSENAK-
KLAKEVEN), c-Myc (PAAKRVKLD) and TUS-protein (KLKI- 5.1.3. Electroporation
KRPVK)� represent some examples of commonly used NLESlse ctroporation is the application of an external electrical field that
(Fortier et al. 2014). halts the cell membrane permeability temporarily, allowing the
passage of any molecule oY( ung and Dean 2015 .) It is a simple,
easily applied method that has undergone, in many studies, some
4. Cytotoxicity face-off: Non-viral vs. viral systems
modifications to provide considerable transfection efficiency .
One of the main reasons for the existence of non-viral gene
Moreover, electroporation can be applied to all types of cells and
delivery carriers is their reputation as being safer than the ill-re-
tissues . oH wever, electroporation varies in its transfections for
puted viral vectors. In spite of reputation, non-viral carriers still
each type . As in the case of particle bombardment, electroporation
have their share of toxicity. Aside from barriers and internaliza-
may cause tissue damage in humans oY( ung and Dean 2015.)
tion issues, cytotoxicity of some used materials in non-viral gene
delivery remains an obstacle that needs further investigation .
Toxic effects of gene delivery reagents are mainly due to 5th.1e.i4r. Sonoporation
cationic nature. Interaction between cationic polymers or lipids
Similar to electroporation, sonoporation is forming a pore in the
with the outer and inner cell membranes alter the integrity of
cell membrane transiently via the application of ultrasound . This
these membranes (Juliano and Carver 2015). In some cases,
allows the target molecules to enter the cell O( N’ eill and Li 2008 .)
simply adjusting the molecular weight may reduce polymer
Many studies conveyed an enhanced gene transfer using ultra-
toxicity. In the case of PEI polymer where high MW PEI (greater
sound-assisted microbubbles R( ahim et al . 2006 .) oH wever, many
than 25,000 Da) was found, it was seen to be toxic to cells while,
factors may impede the use of ultrasound-mediated gene delivery .
polymers having medium to low MW (5,000–25,000 Da) were
These factors include tissue damages and that there is no known
more efficient and less toxic. Moreover, nanoparticles have the
dose or limits to the areas affected by the ultrasound waves R( ahim
ability to efficiently transfect post-mitotici nc evilvlso and in
et al . 2006.)
vitro with no or low toxicity and are inert to immune responses .
Finally, hybrid systems can always be the solution to many
5.1.5. Laser irradiation
problems in terms of toxicity and efficacy. Combining different
types of viral and non-viral carriers can lead to an advancedL anseewr irradiation is applying pulsed laser waves using a laser source
system that may conquer many barriers (Fortier et al. 201s4u) . ch as yttrium-aluminum garnet or argon ion or any other laser
Finally, non-viral carriers may have a better safety profile soovuer ce . Therefore, a pore in the cell membrane is formed allowing
viral vectors, but this does not mean that they do not showpl aasnmy ids to be transfected from the medium to the cytosol of the
toxicity. On the contrary, they may be as toxic and problemcaetlilc T( ao et al . 198 .)7 It was first studied in 1986 using neodymi-
as viral vectors. The key advantage of non-viral vectors isu tmh-ed oped yttrium aluminum garnet K( urata et al . 1986 .) The laser
versatility in its design and modulation, enabling researcheirrsa dtioa tion method showed substantial transfection efficiency . The
630 Pharmazie 72 2( 01)7
REVIEW
niatrec ta slec eht ot suodrazah eb osla yam noitaidari resal fo esu TA newteb ecneref fid eht ,elpmax e ro F .sdohtem P )e v isnopseroib(
snoitartnecnoc ataru K( te .la 1 .)689 , ylaniF resal noitaidari si snoitartnecnoc ni eht ralulecartx e dna ralulecartni secirtam fo a
.etad ot refsnart eneg rof naem elbaiv a deredisnoc TA teg lec rof nac gniledom eb na a rat e remylop oM( v P isnopser
te .la102 esre v A elucelomoib id .)4 nac tca sa a teg rat rof -igoloib
TA e ,sdohtem hcus sa v isnopser-ylac P dna semyzne gnauH( te .la
5.2. Chemical carriers (inorganic particles, synthetic/
102 R ;3 N dna htromama 102 rake vra .)5
natural lipids, polymers, and peptides)
T sih puorg fo sreirac sedulcni dipil dna desab- remylop ,srotce v
6. Cargoes delivered by non-viral carriers
ni noitida ot selcitraponan dna .seditpepC lacimeh srotce v e v ah
tneref fid selur gnidulcni noitcetorp fo citeneg lairetam morf I n , lariv-non citeneg eneg yre v lairetam iled derefsnart si ylniam
,noitadarg ed gnire v iled ti otni teg rat ,slec gnicnahne eht reirac dei fisalc otni eht gniw olof sepyt fo cielcun sdica xuodiM( te .la
ytefas ,eli forp deniatsus esaeler ro/dna gnicnahne noitcefsnart :)902
R( y caci f fe N dna htromama 102 rake vra N .)5 lacimehc/dica cielcu • OA( seditoelcunogilo esnesitnA N-ehtnys selucelom detaler ro )
oitarN( P/ oitar ni esac fo ,sremylop erehwN setonedN negorti no ylacimehc dezis
eht remylop enobkcab dna P setoned eht srohpsohP no ehtND A • ND eg raL ND dimsalP( selucelom A NDp ;A )A
,)enobkcab noitulos ,Hp dna lec enarbmem snoitidnoc era la • RNI A Ris( ecnerefretn NRim ,A NS ,A Rh N)A
srotca f taht tcef fa eht noitcefsnart y cneici f fe fo a desab-lacimehc
yre .dohtemCviled lacimeh sreirac yler no rieht cinoitac erutan ot
6.1. Antisense oligonucleotides (AON)
deg cielcun sdica yle ouG(rahc dna v ita g x eht yltneici en elpmoc f fe
,t s r i F na es n e s i t n a ed i t o e l c u n o g i l oOA( N ) si a ci t e h t n y s - e l g n i s
102 gnauH E dna miK ;2 102 eniwreb .)0
de d n a r t s ,e d i t o e l c u n o b i r y x o e d hc i h w si 1 03–5 se d i t o e l c u n gn o l
C( OA .)6 0 0 2 . l a t e n a h Nv gni c n e l i s e n e g e s u a c ia eht h t i w g n i r e f r e t n i
5.2.1. Lipid and polymer T.ni e gt n o i r s p u a c - n e o s i f a t o e e c h s u t i d d o r p t e g ye s r u o i r f t e a e h t h t
R reg n e s s e m Nv tse r r a l a n o i t a l s n a r t g n i s u a c , A ia fo e c n a r d n i h c i r e t s
-a g desab- sreirac nac ylreporp esnednoc eht en dipiL dna remylop
R fo n o i t c u d n i d n a y t i v i t c a l a m o s o b i r N yti v i t c a e s a e l c u n o d n e H e s a
yle v it deg rahc ND eud ot rieht hgih ytisned fo e v itisop seg rahc
C( na h t e . l .a ) 6 0 0 2SO noit c e fl A o e e h t N ec n - e a u r d q o n e p a s r o c n i
I( meharb te .la 102 .)4 noitaraperP dna yticixot fo desab-dipil
OA eh t n i s e d i t o e l c u n d e i f i d o m - y l l a c i m e h c n i a t r e c f o n o i t N yle g r a l
detroper stcef fe cixot emos htiw ,erutcurts rieht no dneped sreirac
t c e f f a s t i m s i n a h c e m f o n o i t c a d n a e h t t n e t x e f o g n i t a l u d o m e n e g
rehcsiF( te .la .)302 T ereh era lare v es sepyt fo desab-dipil
no i s s e r p xy e e v a e l e D ( dn a ah m a D 102 .) 2 ,e r o m r e h t r u F en oOA N
,sreirac fo hcihwOD S AN-yxoly eloid-3,2( P -xobracenimreps(2[-
s u r i v o l a g e m o t y c t a e r t o t n o i t a r t s i n i m d a l a c o l r o f d e v o r p p a n e e b s a h
N-]ly hte-)odima N, 1-lyhtemid- muinimanaporp- oroulfirt ,)etateca
®
OD T AM N( 1[- )yxoly oeloid-3,2(- N-]ly porp N, N, -omay htemirt- n e s r i v i m o F ( ,1 ;89 9 e n o l l i r G d n a z n a L1002 ;n a m y e l h g i H1 ,a8 9 9
muin ,)edirolhc dna semosopil era eht tsom tnatropmi .selpmax e , ) b e l i h wO reh t o A N s dn a l l a m s g n i r e f r e t n iRN ARis( N )A er a n i
eneg lariv-non tnatropmi na deredisnoc era ,ci ficeps ni ,semosopiL C( tn e m p o l e v e d l a c i n i l c f o s e g a t s s u o i r a v . ) 6 0 0 2 . l a t e n a h
reirac .metsys I n eht esac fo ,semosopil eht citeneg lairetam nac
eb deta gujnoc ot eht lanretx e eca frus fo eht semosopil ot mrof
6.2. Plasmid DNA
sex elpopil sa ni eht esac fo eht ylaicremoc elbalia v a -tcefopiL
A dimsalp si a ,lams , ralucric lamosomorhcartx e dna -elbuod
™enima rengleF( te .la1 ;59 amayuraM te .la .)702I n siht ,esac
dednarts ND A elucelom taht sref fid morf lamosomorhc ND .A
yle v ita g en eht eta gujnoc ot erutan ni cinoitac eb ot e v ah semosopil
NDp A si ylarutan dnuof ni lairetcab slec dna emos .setoyrakue
deg rahc citeneg .lairetamO n eht rehto ,dnah eht citeneg lairetam
gniruD ,noisiv lairetcab la id fo eht sdimsalp deniatnoc nihtiw eht
nac eb detaluspacne edisni eht lamosopil ralucise v .erutcurts I n
sdimsalp re v iled osla nac airetcaB .slec rethguad ot deipoc era lec
erutan ni cinoinon no ,cinoina ,cinoitac eb nac semosopil ,esac siht
ot eno rehtona via noita gujnoc hsidoL( dna y ksrupiZ102 .)T eh
rengleF( te .la1 .)59 I n ,noitida noitalumrof dna yticixototy c fo
ezis fo a ylnomoc desuNDp A segnar morf w a derdnuhef -esab
desab- remylop sreirac osla dneped no eht dnuopmoc ,erutcurts
sriap )pb( ot lare v es dnasuoht .spb I n noitida ot eht ,enegsnart
htiw ylareneg w ol yticixototy c rehcsiF( te .la .)302 C cinoita
NDp A sniatnoc rehto yrotalug er slangis hcus sa eht , retomorp
sremylop nac eb dei fisalc otni desab-larutan ,sremylop hcus sa
, retroper recnahne ,secneuqes ,gnicilps dna noitaly nedaylop
,nasotihc dna ylacitehtnys ,derutca funam hcus sa -inely hteylop
,setis hcihw e v ah tnatropmi selor ni gnitalug er eneg noiserpx e
EP( enim II( )LP( enisyl-L-ylop dna ) 102 .la te meharb .)4
W( smaili te .la .)902 T eh noitceles fo a elbatius recnahne nac
e v orpmi noitpircsnart y cneici f fe yb lare v es derdnuh sdlof N( so
®
5.2.2. Peptides te .la .)202TefiL edi SW 5V( laminA XG )htlaeH si deredisnoc
eht ylno de v orpa dimsalp ND A rof htw org enomroh gnisaeler
- o p o c s a d e r e d i s n o c e r a t a h t s d i c a o n i m a f o s n i a h c t r o h s e r a s e d i t p e P
nongiM( te .la102 .)5 y naM rehto namuh dimsalp stcudorp era ni
n e t x e e v a h s e d i t p e P . s r e m y l e h t d n a y r e v i l e d e n e g n i s e l o r l a t i v y l e v i s
102 yuqodrohcnA dna uX( slairt lacinilc fo sesahp tneref fid 1.)
S .e d i t p e p f o e p y t e n ie sh yt l n fo o d ,e s ea db i t p e ps e i r a c i t v e h t n y e l o r
r o e n i n i g , e r s a a eb v a h a t a e r g e l o r n ig n i s n e d n o cND A s a k i l u o B (
102 ; 6R da a t e . l a102 . ) 4O r es h e td e ic gt n n p a i e h r p n e e e h v t f o i l e d
6.3. RNA Interference (RNAi) using siRNA, miRNA,
eht c i t e n e gl a i r e t a m o t e h t l l e c r o e h t s u e l c u nR( da a t e . l a 102 ; 4
shRNA
okr o Z d n a l e g n .a ) L 5 0 0 2 n a i Q t e . l a102( ) 2 d e s uTNG ed i t p e p n i
RN A ecnerefretniR( N )iA si a yrotalug er msinahcem taht sruco
t n e r e f f i d h t i w s e d i t pTep f. o g n i t e g s e r p a y t t n r i e a h r t b o y n a m e r a e r e h
ni eht msalpoty c fo tsom citoyrakue slec ot . lortnoc eneg ytiv itca
R( s r e i r r a b l l e c e m o c r e v o n a c t a h t s e i t r e p o r p 102 . l a t e d a a . ) 4
S ralimiO ot A N ,ngised eht reporp noitceles foRis N A ecneuqes
dna noitaroprocni fo niatrec dei fidom-ylacimehc seditoelcun nac
5.2.3. Inorganic nanoparticles
ecnahne sti ytici ficeps dna eneg gnicnelis ytiv itca relw oD( te .la
I cina g ron selcitraponan era rehtona yrog etac-bus fo lacimehc .)602 RN A nac osla eb derefsnart otni slec yb noitaluspacne
srotce v rof eneg . yre v iled T yeh era ylisae deraperp dna e v ah ni ,semosopil ro e kil-dipil selcitraponanNL( )s iL( te .la 102 .)6
etaredom yticixot niJ( te .la 102 .)4 dloG selcitraponanNuA( ,)sP E decnahn yre v iled fo Ris N A se vlo v ni tnela v oc seta gujnoc ot
nobrac sebutonanC( NT ,)s acilis ,selcitraponan dna mutnauq stod gnitartenep-lec seditpepC( )sP ro nietorp noitcudsnart sniamod
.puorg siht fo selpmax e tnatropmi tsom eht gnoma era )sDQ( R dna nauG( 102 re kceneso .)7
5.3. Biologically-responsive methods 6.3.1. Mature micro RNA (miRNA)
segnahc lacigoloib lamron eht ot sdnopser taht reirac a gningiseD Rim N A si a ,trohs 42-02 ,seditoelcun elgnis dednarts gnidoc-non
ni eht namuh ydob si eht lapicnirp fo eht e v isnopser-ylacigoloib RN A taht sdnib ot ylaitrap yratnemelpmoc setis no rieht teg rat
7 eizamrahP 2102( )7 136
REVIEW
mRNA and inhibits mRNA after transcription . miRNA silencl( einse ar and branched polymers, dendrimers and polysaccharides,)
gene expression by affecting the stability of target mRNAs otrh ivoila modified polymers, micelles, biodegradable nanoparticles,
translational repression W( ilczynska and Bushell 2015 .) PEyG lated polymers, cell-penetrating peptide grafted carr-iers, inor
ganic nanoparticles, and others . Furthermore, surveyed patents and
papers also illustrate heterogeneous ain-dvi nvo-v itro evaluation
6.3.2. Short hairpin RNA (shRNA)
methods, physicochemical characterization parameters, transfec-
shRNA is a synthetic RNA sequence that can induce gene silencing
tion tests, expression readout techniques, reporter and therapeutic
via RNAi . It consists of a stem that can vary from 25 to 29 bases
genes, toxicity testing, genetic material doses, cell lines used in
and a loop of 4 to 23 nucleotides eG( et al . 2010.)
testing, animals’ models, therapeutic purpose of non-viral delivery
system, and even the controls or gold standards used in comparing
6.4. Toxicity and immune responses of cargoes delivered their novel carriers with . Such heterogeneity and lack of standard
or even consistent methodology protocols can be drawbacks in the
by non-viral carriers
progress of the field . The complete list of the surveyed patents and
Exogenous genetic materials, DNA or RNA, surely stimulate the
papers is categorized and tabulated in Appendices 1 and 2 .
immune system . This results in release of inflammatory cytokines
In this part of the review, the most common types of non-viral gene
which cause local and systemic inflammatory reactions . Conjuga-
delivery carriers are discussed along with representative examples
tion with cationic lipids or polymers usually increases the immune
from the literature, either published as papers or registered patents
response . Immune reaction and release of cytokines are mainly
over a time-span extending from 2006 into 201 .7
attributed to the endosomal toll-like receptors T( LR ) transduction
K( arik ó et al . 2004 .) The intracellular receptors TLR3, TLR,7
TLR8, and TLR9 are the main receptors involved in the ide7n.1ti.- Cationic polymers
fication of nucleic acid-like structures M( orozumi and eU nishi
7.1.1. Poly-l-lysine (PLL, the safety gold standard)
2009 .) Receptors involved in immune response differ according to
PLL is a synthetic linear polypeptide with repeated l-lysine resi-
the genetic material . The recognition of the immune-stimulatory
dues F( ig�. 1 .) It is one of the most studied cationic polymers in
sequences in the plasmid structure i( e. . unmethylated Cp G motifs )
the field of gene delivery and is considered one of the commonly
is usually mediated via TLR9, which can be reduced by decreasing
used gold standards with respect to safety L( in and Lou 2012 .)
the Cp G content of the pDNA . RNAs are generally recognized by
PLL contains only primary amines, lacking any secondary or
three main types of immune-receptors: TLR 3( , 7 and 8,) protein
tertiary amines . This provides PLL with its high safety profile,
kinase R, and helicases K( arik ó et al . 2004 .) The incorpora-
but drastically affects its transfection efficacy L( in and Lou
tion of few 2- O-methyl modified bases into highly immune-stimu-
2012 .) PLL is responsible for the DNA condensation . The good
latory siRNA was found to evade the induction of the inflammatory
binding of PLL to DNA is due to electrostatic attraction between
cytokinesin vivo uJ( dge et al . 2006 .) In case of AON, immune
its positive charges and the negatively-charged phosphate group
response occurs only if Cp G sites were found on the AON sequence
D( NA ) P( atil et al . 2011 .) Targeting of the malignant cells may
and is due to Toll-like receptor 9 T( LR9 ) mechanism.
be achieved through the electrostatic attraction between PLLs’
positive charge and negatively-charged malignant cell surfaces .
6.5. Advantages and disadvantages of cargoes delivered
On the negative side, complexes of PLL and DNA do not easily
by non-viral carriers dissociate, making PLL of fair transfection efficiency compared
to other polymeric carriers . Moreover, nonspecific interactions
A main advantage for the use of non-viral gene delivery systems is
with cell membranes affect its cytotoxicity L( in and Lou 2012 .)
their ability to carry large-sized DNA genetic material as compared
to the limited packaging capacity of viral carrier systems T( hoTmhearse fore, to overcome PLL weak transfection efficiency, PLL is
et al . 2003 .) When it comes to the type of genetic material, mRNaAlw ays found in a di-block or tri-block form with other polymers
targetingvi a antisense therapy A( ON, siRNA, shRNA and miRNAt ) o balance the criteria of the delivering vector . Also, PLL is usually
was found to have many privileges over pDNA . Less amountfso uonf d PEyG lated or modified with imidazole groups to enhance
the delivery vehicle are usually required . This leads to reductiiotsn itnra nsfection and toxicity profiles L( in and Lou, 2012 .) uY et
cost and cytotoxicity . In addition, the vector is easily designeda,l .a2( n0d0 )7 designed PLL-grafted chitosan copolymer to enhance
the nuclear barrier and insertional mutagenesis can be escatpraends ifne ction and reduce cytoxicity in vivo . The good DNA binding
contrast to plasmid DNA K( utzler and Weiner 2008.) of PLL and biodegradability and compatibility of chitosan cause
Nevertheless, the nuclear barrier can be a problem for -someP tLhLe-rgrafted chitosan copolymer to yield better transfection
apeutic applications of AON and the inability to express the egfefincaec y compared to that of each polymer individually uY( et al .
of interest is a drawback of the antisense therapy E( lsabahy 2e0t 1a1l .) . Patil et al . 2( 011 ) developed a cationic PAMAM-PE-G PLL
2011.) nanocarriers� ystem for targeting cancer cells . PLL primary amines
As for plasmid DNA, it can be used to express a missing agnedn eP AMAM tertiary amines provide the advantage of a block
and as an adjuvant therapy in vaccination . oH wever, as previoucsolpyo lymer with good DNA binding capabilities, transfection effi-
mentioned, pDNA has the obstacle of the nuclear barrier, pcoisesnic-y, endosomal escape, and cytoplasmic delivery of the genetic
bility of insertional mutagenesis, and difficulties in formulatimnga tiet rial P( atil et al . 2011.)
K( utzler and Weiner 2008.)
7.1.2. Polyethyleneimine (PEI, the effciency gold stan-
7. Carriers from the early synthetic polymers to the dard)
latest smart polymeric modifcations
PEI is a three-member ring of ethyleneimine that, in the presence
Over the past three decades, there is a wave of revolution oof fn ae wca talyst, polyethylenimine (PEI) is formed (Fig1.� ). Linear or
strategies for non-viral gene delivery iY( n et al . 2014 .) More thanb ranched PEI is characterized by high-charge density and proton
50 patents and 90 research papers describing novel strategsiepso nfogre effect (Liu et al. 2011b). Behr et al. were the first to detail
delivering plasmid to target cells have been screened and thtaeb us- e of PEI in non-viral gene delivery in 1995 (Boussif et al .
lated in Appendices 1 and 2 . This literature-extensive scan p1r9e9s5-). Their paper was a key milestone in the history of non-viral
ents a� time-span� extending from 2006 into� 201 .7 The bold andg ene delivery technology. Prior to it, none of the available non-viral
®
revolutionary introduction and marketing of eG nd inci n2e003 gene delivery carriers ever matched the efficacy of viral vectors
from China fueled the gene therapy field and fed confidencetr ansdf ection efficiency. Behr et al. demonstrated strong transfec-
encouragement to startups and researchers worldwide to folltoiown- uepff icacy with PEI empowered by its buffering capacity and its
and lead the field in the Western world . Screened patents parnodto n sponge effect coupled with high cationic charge imparted
research papers illustrate different novel vectors such as poblyym ietsrs u nique primary, secondary, and tertiary amine groups in
632 Pharmazie 72 2( 01)7
REVIEW
1 .giF C : .smetsys reirac eneg lariv-non fo noitalumrof eht ni desu sdipil dna sremylop suoremun fo erutcurts lacimeh
i t s st r u c t u r e ba c k b o n e (B o u s s i f et al . 1995) . Since th e n , PEI i s iL te .la 102( )4 dengised a yranret x elpmocEPm -s(-g-LP-b-G
c o n s i d e r e d t h e g o l d s t a n d a r d i n t h e n o n - v i r a l g e n e e r yv d e o l r w i l d EPl I ) ni a namuh naira v o recnac . y parehtWML EP I sa w detfarg
T. wi t h r e i s yc p a e cf c t t o e f h e p r o t o n s p o n g e yh p o t h e s w i s t s s h h a o t ot aEP LP-G remylopoc gnisu eht edi flusid egaknil ot e v orpmi
u n p r o t o n a t e d am i n e s in s i d e th e ly s o s o m e s at t r a c t pr o t o n s ; an d , Ris N A noitcefsnart dna redner eht remylopoc elbicuderoib dna
a s a r e s u l t , t h e r e i s a n i n c r e a s e i n t h e i n f l u x o f c h l o r i d e i o n s a n d .elbadarg edoib T eh elbicuderoib erutaef selbane eht reirac
, t h e r e p u . F l i s n i a o l n b l e y t a w tw e e e r n p r o t o n s a n d t h a e t w e r i n f l u x metsys ot eb dilos dna tcatni ylralulecartx e dna ot etarg etnisid
l e a d s to sw e l l i n g an d ur s bt s of ly s o s o m e s (B e n j a m i n s e n et al . ylisae ni eht elbicuder tnemnoriv ne . ylralulecartni ,eromrehtruF
2 01Al3t) h . o u g h PEI is c o n s i d e r e d t h e g o l d s t a n d a r d i n n o n - v i r a l y eht deta gujnoc a lanolconom ydobitna )nitpecreh( taht stca sa a
g e n e er yv d el i w h e n it co m e s to , iyc a cf e f i t alf l s sh o r t wh e n i t gniteg rat dna gil htiw EP LP-G ot ecnahne noitcefsnart y caci f fe
c o m e s t o s a f e t y a n d t o x i c i t y p a t t e r n . , P r a c t i c a t l h l e y m a i n r e a s o n niS 3-v o k lec( enil fo naira v o .)recnacEPm EPl-s(-g-LP-b-G I )
t h a t a c c o u n t s f o r PEI iyc acf e f ( h i g h e pvo s i t i c h a n g e c o n t e n t ) i s decuder eht yticixototy c eud ot EP I notorp gniref fu b ytiliba dna
a l s o t h e s o u r c e o f i t s h i g h t o x i c i t y ip lr eo . f PEI can b e c l a is es di f 102 .la te iL( ytilibadarg edoib LP .)4
a c c o r d i n g t o i t s s t r u c t u r e i n t o l i n e a r a n d b r a n c h e d . A n o t h e r c l a s - dehcnarB EP I si deredisnoc eht dradnats mrof fo eht . remylop
i cs ai tf i o n i s a c c o r d i n g t o i t s m o l e c u l a r w e i g h t . A c c o r d i n g t o PEI dehcnarBEP I , dna yraitret ,senima , yradnoces stsisnoc fo yramirp
m m o o l l w e e cc lu u w b l la l e i h c a e o as ia i n i a n rs re gt i g n d id ho t h f t , la fo hcihw nac eb .detanotorp T ,erofereh ti si fo elbaredisnoc
w e i g h t PEI. w Lo m o l e c u l a r w e i g h t ( L MW) PEI has w e r l o y t oc - gniref fu b yticapac dna e v itisop eg rahc tnetnoc dna yltneuqesbus
t o x i c i t y t h a n t h a t o f h i g h m o l e c u l a r w e i g h t ( H MW) PEI an( gF e t sah a reteb noitasnednoc foND A naht taht fo raenilEP I . etipseD
a l . 2 014). , e r v w e H o d u e t o i t s w l o g e n e t r a n s f e c t i o n , L MW PEI sti hgih noitcefsnart , y cneici f fe yticixototy c fo dehcnarb EP I si
i s m io ed di f b y a yh d r o p h o b i c g r o u p t o i n c r e a s e i t s g e n e t r a n s f e c - a rojam egnelahc sa EP I yticixot sesaercni htiw gnisaercni eht
t i o n . i c i fye n c e f PEI gra f t e d c o p o l y m e r s u s i n g d i is du el f l i n k a g e s 102 .la te uiL( gnihcnarb ro thgiew ralucelom 1.)a
a r e en p vr o t o en h a n c e ge n e tr a n s f e c t i o n an d ge n e r a t e a sm a r t elbadarg edoiB edi flusid segaknil dna retse spuorg era deda ot
b i o r e s pv o n s i a n d b i o r e d u c i b l e n o n - v i r a l g e n e c a r r i e r a r( hP i z e t EP I ot esaerced sti . yticixototy c e kiL y nam rehto cinoitac -ylop
a l . 2013). On the ot h e r ha n d , HMW PEI xhie b i t s a hi g h DN A ,srem EP I nac eb EP detalyG ot ecnahne sti yticixototy c eli forp
H MWU,n f o r t u n a t e l y . i c ti rfye an nc se f e c t i o n PE Iytoc - h i g h a h a s eud ot enely hteylop locylg gnidleihs tcef fe gna Y( te .la 102 .)5
t o x i c i t y d u e t o i t s n o n -g dr ea d a b l e l i en rk s . S emo ,stnetap dehsilbatse rof EP I , dew ohs taht EP I nac eb desu
7 eizamrahP 2102( )7 36
REVIEW
in chemotherapy and radiotherapy treatment . It showed promPisLiAGng-b ased nanoparticles were tested in miRNA-related diseases
potential as a polymeric carrier system to be used therapeustiuccahll ya s diabetic, cardiovascular, and neurodegenerative diseases
and as a diagnosis tool C( hrony et al . 2010 ; Lynn and Miller 2012L( .) iang et al . 2011 .) Biodegradable PLAG developed higher gene
inhibition efficiency than Lipofectamine in a study performed to
treat lung cancer D( u et al . 2012.)
7.1.3. PAMAM (Poly(amidoamine) dendrimer)
Dendrimers have emerged in 1995 with a final d‘ endrimer-like
7.1.5. Chitosan (the natural polymer)
star-branched polymers ’ shape iH( rao and oY o 2011 .) They have
a unique three-dimension branched-architecture . Their nano-Csihzietods an is a biodegradable cationic polymer that has free amino
structure and cationic charge made them well suitable as promacisidinsg t hat can bind to negatively-charged moieties such as nucleic
candidates for non-viral gene delivery . Dendrimers are syntheascizideds P( atel et al . 2010 .) It is composed of N-acetylglucosamine
by capping the chains of a hydroxyl terminated six arm star poalynmd egrl ucosamine F( ig1�. ) and can be obtained by alkaline deacetyl-
with dendrons r( ing-opening .) These produced m“ acro-initiaa-tion of chitin . Chitosan is found to be nontoxic, biocompatible,
tors�” are polymerized to produce the D“ ENDRIMER ” polymer and biodegradable . Its degradation products by lysosomes are
iH( rao and oY o 2011 .) Polya( midoamine ) P( AMAM ) dendrimers nontoxic, non-immunogenic, and non-carcinogenic . Due to its
are hyper-branched polymers with molecular uniformity, nanrraotuwr al origin, chitosan and its derivatives present significant phys-
molecular weight distribution, defined size and shape chiacroacch-emical and structural variability, leading in high variability in
teristics, and a multifunctional terminal surface . The PAMApMha rmaceutical formulations E( lsabee and Abdou 2013 ; Ili’ na and
dendrimer polymer was described by Tomalia et al . 1( 985 ) as s“ taVra-rlamov 2005 ; Patel et al . 2010 .) Chitosan has wide applications
burst ’ ’ dendritic macromolecule . PAMAMs’ design is based oin the field of non-viral gene therapy due to its biocompatibility
spherical structure based on a sphere-like core and is characatnerdi zleodw toxicity . oH wever, it has low transfection efficiency l( ow
by an internal molecular design consisting of tree-like brancehnidnogs, omal escape ) A( kbuga et al . 2016 .)
with each external l‘ ayer,’ or generation, containing exponentiMalulym per et al . introduced chitosan as a gene delivery carrier in
more branching points . Based on this design language, PAM1A99M5 C( entelles et al . 2008 .) Chitosan has reactive sites for ligand,
dendrimers may commercially exist in different forms of branchoinnjgu gation, cross-linking, and modification by acetylation of the
or, what is called, different generations F( ig�.1 .) PAMAM possesgs lucosamine monomers, affecting the charge and changing the
cationic primary amine groups at the surface . Such structurew eaigdhs t of the carrier system . These modifications can enhance
in efficient binding with DNA and siRNAs and increases ththeeir efficiency of chitosan . Large numbers of clinical trials applied
intra-cellular uptake by forming nano-sized complexes cathlle db ioconjugation technology to modify chitosan by adding a
d“ endriplexes ” K( esharwani et al . 2012 .) PAMAM-dendrimer synthetic polymer such as PEI, forming Polyethylenimine grafted
DNA complexes d( endriplexes ) aid proficient cellular uptake cbhyi tosan P( EI-g-chitosan ) D( ang and Leong 2006 .) The main aim
the endosomal escape mechanism as in the case of PEI M( ajoorof s uect h modification is the enhancement of the transfection efficacy
®
al . 2008 .) Dendrimers are commercialized as Supea� rnFdec Ptoly- of chitosan and the toxicity profile of PEI . PEI-g-chitosan-DNA
®
Fect , which are known for high transfection DNA characterisctoicms plex was shown to be stable complex with high transfection
M( assadeh et al . 2015 .) As in the case of PEI, PAMAM dendrimerse fficiency and low cytotoxicity profile D( ang and Leong 2006 .)
suffer from a high toxicity profile . Chitosan has been investigated intensively for non-viral gene
Kim et al . 2( 013 ) designed a bioconjugate system of arginine-grafdtedliv ery . A nanoparticle-chitosan complex was developed to
poly c( ystamine bisacrylamide-diaminohexane ) A( BP ) ando vercome the instability barrier of RNA molecule in biological
PAMAM P( AM-ABP/chimeric DNA polyplex ) to treat type 2 environment R( agelle et al . 2016 .) Furthermore, chitosans’ cova-
diabetes via enhancing of exendin-4 expression . Furthermleonrtel,y -bonded hyaluronic acid nanoparticle was used for ocular
PAM-ABP enhances insulin secretion via activation of prodteisine ases treatment uZ( lliger et al . 2015.)
kinase K P( KA .) In another study, PAMAM-Arg/DNA modifica-
tion was clinically applied for ovarian cancer therapy aJ( ng et al .
7.1.6. PDMAEMA polymer (poly (2-(dimethylamino)-ethyl
2011 .) Furthermore, Kobyashi et al . 2( 016 ) used carbonyl reduc-
methacrylate)
tase 1 c( br1 ) DNA with a polyamidoamine dendrimer P( AMAM )
to target ovarian cancer . In another example, Ayatollahi etP DalM . AEMA is a water soluble polycation F( ig1�. ) S( ynatschke et al .
2( 015 ) designed PEyG lated alkylcarboxylate-grafted PAMAM 2011 .) It can be easily synthesized by atom transfer radial polym-
dendrimers 4G( and 5G ) which had a high transfection efficacye rization technique A( TRP .) It has a high transfection efficiency
in mesenchymal stem cells M( SCs ) and murine neuroblastobmecaa use it has the ability to escape from endosomes in the same
N( euro-2a ) celll� ine A( yatollahi et al . 2015 .) fashion as PEI S( ynatschke et al . 2011 .) Its cytotoxicity depends
on the molar ratio of its co-polymeric subunits and the polymer
concentration . As the NP\ ratio increases, the cytotoxicity decreases
7.1.4. Poly lactic-co-glycolic acid copolymer (PLGA)
iQ( an et al . 2012 .) On the contrary, as the polymer concentration
PLAG is a biodegradable polymer that is used as a controlled dinrucrge ases, the cytotoxicity increases R( awlinson et al . 2010.)
delivery carrier F( ig1�. .) PLAG has been approved by the SU FDA As for many polycations, having a net positive charge on their
for the use of drug delivery, diagnostics, and other applicaotuiotenrs- structure, the positive charge enhances the DNA condensa-
L( u et al . 2009�.)For this reason, PLAG is considered a gold stan-tion and encapsulation into the polyplex . oH wever, increasing the
dard with respect to safety . Furthermore, due to its copolympeorsiicti ve charge increases the cytotoxicity due to non-sp-ecific inter
nature, varying the content of polylactic acid or polyglycolica acctiiodn with the negatively-charged cellular compartments as RBCs
in PLAG enables researchers to control the rate and extenta nodf proteins S( ynatschke et al . 2011 .) Furthermore, the higher
biodegradability of PLAG L( u et al . 2009 .) molecular weight of PDMAEMA leads to a higher transfection
PLAG may be formulated as nanoparticles which can protect dreuffgicsa cy and cytotoxicity S( amsonova et al . 2011.)
from degradation and enhance their stability D( anhier et al . 20I1n2 .)studies with several polymers, polyethylene glycol has always
PLAG -based nanoparticles can improve phakrminaectioc and been known for its shielding effect . PE G increases the relative
pharmacodynamics profiles D( anhier et al . 2012 .) Liu et al . 2( 012 ) colloidal and serum stability of PDMAEMA, prolongs the circula-
designed mPE-G PLAG -b-PLL amphiphilic block copolymer tion time, and prevents protein adsorption into aggregates K( halil
for the delivery of adriamycin and siRNA for hepatic carcineotm al . 2006 .) Furthermore, PE-G PDMAEMA complexes have
treatment . Adriamycin-mPE-G PLAG -b-PLL or siRNA-mPE-G reduced cytotoxicity compared to PEI due to lower surface activity
PLAG -bPLL nanoparticles showed negligible cytotoxicity ando fa t he complex iQ( an et al . 2012.)
high compatibility with live cells L( iu et al . 2012 .) PLAG -based Moreover, addition of surface ligands to enhance targeting of poly-
nanoparticles were tested to target nucleic acids to hepato-cmelelursla tro target tissue is a valid option to minimize the chance for
carcinoma with a good safety profile L( iang et al . 2011 .) Moreovenr,o n-specific interactions . For instance, brain targeting has been of
634 Pharmazie 72 2( 01)7
REVIEW
reirab niarb dolb eht ot seca detcirtser ot eud egnelahc rojam a noitanibmoc foOD T PA htiw lartuen repleh sdipil dluohs eb desu
.)B( TNG , a 1 dicaonima-2 niahc taht delae v er a tnaci fingis 102 y ebdoG dna szalaB( y caci f fe noitcefsnart ecnahne ot 1 .)
niarb ,tropsnart sah neb deta gitse v ni ot teg rat niarb slec naiQ( amay e kataH te .la )902( dengised na recnacitna lanoitcnufitlum
te .la102 .)2T eh kcolb-irt x elpylop foTNG EP- EAMDP-G AM si epyt-epole v ne eciv ed-onan EM( N )D deta gujnoc htiw EP -pep-G
nw ohs ot e v ah dog noitasnednoc , yticapac roirepus noitcefsnart OD-edit EP .)DP( T eh eciv ed-onan nac eb de v aelc ni a xirtam
, y dna cneici f fe ylde kram w ol yticixototy c htiw tneici f fe gniteg rat esanietorpolatem hcir-)PM( ,tnemnoriv ne htiw decnahne
ot eht niarb naiQ( te .la 102 .)2 T eh noitcefsnart y cneici f fe fo yre v iled y dna caci f htob fe in vivo dna in vitro amay e kataH( te .la
TNG dei fidom- se x elpylop sa w rehgih naht taht fo dei fidomnu .)902
i htob se x elpylop n vitro i dna n vivo 102 .la te naiQ( .)2
CD C- loh
β3 N[ N(- N,’ ,loretselohc]ly omabrac-)enahteonimaly htemid-’ ro
7.2. Lipid-based carriers
CD C- ,loh sa w dezisehtnys yb oaG dna gnauH ni 119 oaG( dna
desab-dipiL selucelom desu ni eneg y pareht yre v iled era -isalc gnauH119 T .) eh loretselohc yteiom si elbitapmocoib dna .elbats
dei f otni dipil se x elponan elpmis( se x elpmoc fo cinoitac sdipil T ,suhCD C- loh sah a rehgih noitcefsnart y caci f fe )dlof-4-2( naht
dna yle v ita g en deg rahc ND ,)A semosopil rehtie( sa se x elpmoc nitcefopiL ® C( noiserpx e esarefsnartlyteca locinehpmarolhc ni TA
htiw deg rahc-yle v ita g enND A ro ND A detaluspacne nihtiw eht )yasa szalaB( dna y ebdoG102 1 .)CD C- loh secuder eht -xototy c
,)semosopil dilos eroc desab-dipil ,selcitraponan dna desab-dipil nitcefopil ot derapmoc dlof- ruof ot pu ytici ® lec emos ni yticixot s ’
102 .la te euX( snoislume-onan .)5 senil szalaB( dna y ebdoG102 1 .)C noitanibmo fo CD C- loh htiw
OD EP ND dna y cneici f fe noitcefsnart secnahne eud noitaicosid-A
zlohneraB dna madiuZ( eg rahc eca frus x elpopil eht ni noitcuder ot
7.2.1. Lipid complexes (lipoplexes) and liposomes (vesicu-
11 sehguH dna inamjA( noita g erga dna )89 .)9
lar structures) mediated gene transfer
T eh yre v ocsid fo semosahgnab )semosopil( yb mahgnaB deman(
1.2.7 .2. )puorg enimreps( dipil cinoitac tnela v-itluM
retfa )mih no 1 569 sa w a hguorhtkaerb ni le v on lacituecamrahp
®
1 mahgnaB( sreirac-onan 1 .la te mahgnaB ;39 .)b ,a569 OD SenimatcefopiL( A P )
semosopiL era gnilbmesa-fles .smetsys T eh dipil selucelom OD S ,A P N-yxoly eloid-3,2( )odimaxobracenimreps(2[- N-]ly hte ,
ylroiretni sliat cibohpordy h rieht laecnoc ot ylsuoenatnops esrepsid N l-ly htemid- muinimanaporp ,)etatecaoroulfirt si rehtona cinoitac
dna esopx e rieht cilihpordy h sdaeh ni eht suoeuqa .tnemtrapmoc dipil dezisehtnys sa a e v ita v ired fo OD T .AM T eh erutcurts si
C-opil( erutcurts ralucise v reyalib a otni setaruguani ti , yltneuqesno ralimis otOD T AM tpecx e rof a enimreps puorg hcihw si dnuob
)emos htiw a elcitrap ezis gnignar morf 02 mn ot a w ef snorcim via a editpep dnob ot eht cibohpordy h sniahcS( namarahti 102 1 ;
szalaB( dna yebdoG 102 1 .) lamosopiL ND A se x elpmoc era gnahZ te .la102 .)2T eh noitida fo eht enimreps lanoitcnuf puorg
demrof nehw a deg rahc-yle v ita g enND A elucelom si de x elpmoc sdael ot a rethgitND A noitcapmoc eud ot noitcaretni newteb sti
htiw a emosopil .erutcurts lamosopiLND A se x elpmoc era -emos negordy h sdnob dna ehtND A sesabS( namarahti102 1 ; gnahZ te
semit ylne kats