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Polyethylene glycolgrafted polyethylenimine used to ...

Polyethylene glycol grafted polyethylenimine used to enhanceadenovirus gene deliveryKumar Singarapu, Ivy Pal, Joshua D. RamseySchool of Chemical Engineering, Oklahoma State University, Stillwater, OklahomaReceived 17 August 2012; revised 5 October 2012; accepted 9 October 2012 Published online 27 November 2012 in Wiley Online Library ( ). DOI: :An improved adenoviral-based gene delivery vectorwas developed by complexing adenovirus (Ad) with a biocom-patible, grafted copolymer PEG-g-PEI composed of polyethyl-ene glycol (PEG) and polyethylenimine (PEI). Although an Ad-based gene vector is considered relatively safe, its native tro-pism, tendency to elicit an immune response, and susceptibil-ity to inactivating antibodies makes the virus less than goal of the current study was to determine whether Adcould be complexed with a PEG-g-PEI copolymer that wouldenable the virus to transduce cells lacking the Ad receptor,while avoiding the issues commonly associat

Polyethylene glycol–grafted polyethylenimine used to enhance adenovirus gene delivery Kumar Singarapu, Ivy Pal, Joshua D. Ramsey School of Chemical Engineering, Oklahoma State University, Stillwater, Oklahoma

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Transcription of Polyethylene glycolgrafted polyethylenimine used to ...

1 Polyethylene glycol grafted polyethylenimine used to enhanceadenovirus gene deliveryKumar Singarapu, Ivy Pal, Joshua D. RamseySchool of Chemical Engineering, Oklahoma State University, Stillwater, OklahomaReceived 17 August 2012; revised 5 October 2012; accepted 9 October 2012 Published online 27 November 2012 in Wiley Online Library ( ). DOI: :An improved adenoviral-based gene delivery vectorwas developed by complexing adenovirus (Ad) with a biocom-patible, grafted copolymer PEG-g-PEI composed of polyethyl-ene glycol (PEG) and polyethylenimine (PEI). Although an Ad-based gene vector is considered relatively safe, its native tro-pism, tendency to elicit an immune response, and susceptibil-ity to inactivating antibodies makes the virus less than goal of the current study was to determine whether Adcould be complexed with a PEG-g-PEI copolymer that wouldenable the virus to transduce cells lacking the Ad receptor,while avoiding the issues commonly associated with PEI.

2 Acopolymer library was synthesized using 2 kDa PEG and eitherlinear or branched PEI (25 kDa) with a PEG to PEI grafting ratioof 10, 20, or 30. The results of the study indicate that PEG-g-PEI/Ad complexes are indeed able to transduce CAR-negativeNIH 3T3 cells. The results also demonstrate that the PEG-g-PEI/Ad complexes are less toxic, less hemolytic, and more appropri-ately sized than PEI/Ad Wiley Periodicals, Biomed Mater Res Part A: 101A: 1857 1864, Words:gene delivery, hybrid vector, adenovirus ,polyethylenimineHow to cite this article:Singarapu K, Pal I, Ramsey JD. 2013. Polyethylene glycol grafted polyethylenimine used to enhanceadenovirus gene delivery.

3 J Biomed Mater Res Part A 2013:101A:1857 therapy, which is emerging as a feasible method totreat diseases and has broad potential,1 3is the treatmentof human diseases by transfer of genetic material into apatient s cells. The field had been hindered significantlybecause of difficulty in delivering genetic material in both asafe and efficient manner. Traditional gene delivery vectorsare classified generally as either viral or nonviral vectors are efficient, often capable of infecting dividingand quiescent cells, and can provide long-term gene expres-sion; viral vectors, however, possess drawbacks such as im-munogenicity, pathogenicity, a natural tropism that makestargeting difficult, and a high cost of production and purifi-cation.

4 In contrast, nonviral vectors are relatively inefficient,transfect only dividing cells, and are limited to transientexpression; nonviral vectors, however, possess advantagessuch as low toxicity, immunogenicity, and vectors are also relatively easy and inexpensive toproduce. Because of the above drawbacks, however, neitherviral nor nonviral vectors are ideal gene delivery vector would possess advantagescommonly associated with viral and nonviral vectors andnone of the drawbacks. Improvements along these lineshave been achieved by using both viral and synthetic mate-rials. For example, other groups have worked with a combi-nation of retrovirus and polymers, such as polyethylenimine (PEI),4poly-L-lysine5or ,7 Researchers have alsoused Polyethylene glycol (PEG) to PEGylate or modify thesurface of adenovirus (Ad), which resulted in improvementsto the delivery 10 The approach presented in this study explores the bene-fits of a hybrid PEI/Ad vector.

5 Ad was chosen as the viralplatform for the hybrid vector because the virus is highly ef-ficient, easy to produce in high titers, and virus is also able to transduce dividing and is used currently in more than 23% of theongoing clinical Ad is promising, there aresome serious drawbacks that limit widespread use of the vi-rus. The major drawbacks are immunogenicity and issuesassociated with 18 The polymer PEI, which is the most commonly usedpolymer in gene delivery,19,20assists in cellular uptakethrough electrostatic interactions and promotes studies, however, have reported undesir-able characteristics such as cytotoxicity and incompatibilitywith the circulatory ,22 These studies demon-strated that PEI is not ideal for standalone gene 26To overcome these drawbacks associatedwith PEI, researchers have synthesized PEI copolymers suchas PEG-grafted-PEI (PEG-g-PEI).

6 22 One advantage of using aPEG-g-PEI is that the copolymer is less addition,a PEG-g-PEI/Ad complex will be almost neutrally charged,Additional Supporting Information may be found in the online version of this to:J. D. Ramsey; e-mail: WILEY PERIODICALS, reducing interactions between the complex and pro-teins within the circulatory system. PEGylated copolymersalso elicit less of an immune ,27On the basis ofthese previous studies, a PEG-g-PEI copolymer may be ableto overcome drawbacks associated currently with the Ad/PEI vector while maintaining many of the advantages associ-ated with the virus, such as high efficiency and infection ofquiescent long-term goal of this work is to evaluate the per-formance of a hybrid vector in terms of immunogenicityand targeting flexibility.

7 The focus of the current study wason synthesis of the copolymer and optimization of transduc-tion efficiency on a coxsackie and adenovirus receptor(CAR) deficient cell line. This work also focused on howgrafting ratio (GR) and PEI ( , linear versus branched)affected the polymer characteristics, interactions with serumproteins, toxicity, hemolysis, particle size and charge, andbuffer AND METHODSC ellsHuman embryonic kidney (HEK 293) and mouse fibroblast(NIH 3T3) cell lines were purchased from American TypeCulture Collection (ATCC, Manassas, VA). HEK 293 cellswere cultured in Dulbecco s modified eagle s medium(DMEM) (Gibco-BRL, Grand Island, NY) with 10% fetal bo-vine serum (FBS) (Mediatech, Manassas, VA), and NIH 3T3cells were cultured in DMEM with 10% calf serum (CS)(Mediatech).

8 HEK 293 and NIH 3T3 cells were subculturedevery 2 3 days and were stored in a humidified incubatorat 37!C and 5% and Purification of VirusAd lacking E1 and E3 genes and encoding thelacZreportergene was amplified by infecting HEK 293 cells in 10-cmdishes. The infected cells were incubated until the cyto-pathic effect was observed after which three freeze/thawcycles were performed to lyse the virus-producing cells. Adcontained in the cell lysate was isolated and purified usinga Vivapure Adenopack purification kit (Sartorius StedimGmbH, Heidelberg, Germany). The viral titer (cfu/mL) wasdetermined by infecting HEK 293 cells with serial dilutionsof the virus and staining the infected cells using of PEG-g-PEIOne milliliter of mMPEI (molecular weight [MW] 25kDa) (Polysciences, Warrington, PA) was mixed with variousvolumes, ranging from 90 to 180lL of 5 mMmethoxy PEGsuccinimidyl carboxymethyl (mPEG-SCM, MW 2 kDa) (Lay-san Bio, Huntsville, AL).

9 The mixtures were stirred at roomtemperature for 45 min, after which 50lL of 1 Mglycinewas added to quench the reaction. The reaction mixturewas dialyzed using a dialysis cassette with a MW cutoff kDa. Dialysis was performed in a phosphate-buffered sa-line (PBS) solution containing 5 mMethylenediaminetetra-acetic acid for 24 h and then in distilled water for 48 h. Thedialysate was lyophilized and stored at"80!C. The MW,composition, and GR of the PEG-g-PEI copolymers weredetermined from the MW values of the homopolymers, PEI,and PEG, and H-NMR spectroscopy, where peak areas weredetermined for the -CH2CH2NH"protons of PEI and the"CH2CH2O"protons of PEG (calculations described in theSupporting Information section).

10 Formation of Polymer/Virus ComplexesAd was diluted into serum-free DMEM to produce a desiredmultiplicity of infection of 10. The virus solution was vor-texed while the polymer solution was added dropwise. Theamount of polymer added to Ad was varied from 6 to 150lgof equivalent amounts of PEI for each of the polymers synthe-sized. The polymer/Ad complexes were formed by incubatingfor 10 min at room temperature. The negatively charged Adand positively charged polymer form electrostatic EfficiencyThe transduction efficiency of polymer/Ad complexes wasstudied on CAR-negative NIH 3T3 cells, which are notinfected by Ad alone. The cells were seeded 24 h beforetransduction in 12-well plates, at a seeding density of 1#105cells/cm2.


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