plasmid dna microgels for a therapeutical strategy combining the delivery of genes and anticancer...

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Plasmid DNA Microgels for a TherapeuticalStrategy Combining the Delivery of Genesand Anticancer Drugs

Diana Costa,* Artur J. M. Valente, M. Graca Miguel, Joao Queiroz

Plasmid DNA (pDNA) is encapsulated into biocompatible microgels by an inverse microemul-sion polymerization method. Plasmid DNA and doxorubicin are successfully released frompDNA microgels and their release profiles are characterized by appropriate release models. Theco-delivery of genes and drugs from the microgelsis evaluated as an enhancer of clinical treatment.Moreover, the release of the encapsulated pDNA iscapable of transfection in vitro resulting in theexpression of p53 protein. As a whole, a novelpDNA-based system is described that may findbiomedical uses, especially in the cancer treat-ment through the combined action of chemother-apy and gene delivery approach.

1. Introduction

The evolution in the design and preparation of hydrogels,

namely at the micro and nanoscale, has a great deal of

interest in biomaterials science because of their tunable

chemical and physical three-dimensional structure, good

mechanical properties, high water content, and biocompat-

ibility to tissue and blood. These characteristics give

potential value for the use of hydrogels in biomedical

implants, tissue engineering, bionanotechnology, and

drug/gene delivery.[1–5] Concerning this last issue, a major

research thrust in the biochemical/pharmaceutical techno-

logy is still the development of efficient and safe controlled

release systems for the sustained delivery of drugs and

bioactive agents. To be used therapeutically, these systems

should be able to deliver the drug and/or gene at a specified

D. Costa, J. QueirozCICS – Centro de Investigacao em Ciencias da Saude, Universidadeda Beira Interior, 6201-001 Covilha, PortugalE-mail: [email protected]. J. M. Valente, M. G. MiguelDepartment of Chemistry, University of Coimbra, Coimbra,Portugal

� 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlin

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rate and time period ensuring that the concentration of

the biopharmaceutical in the body would be kept in

the desirable range for a prolonged time. Additionally,

the carrier can be targeted to a particular cell type or an

organ;[6–8] not only protein drugs would benefit from a

targeted delivery to their site of action, but also highly toxic

ones such as anticancer drugs. Concerning drug diffusion,

polymer networks show interesting properties, such as,

ionization of the gel, the extent of swelling, and specific

mesh size. In order to engineered appropriate delivery

systems, mathematical models appear as useful tools to

correlate material properties, interaction parameters, kinetic

events, and transport behavior within complex hydrogel

systems.[9–11] Among the several physical/chemical proper-

ties of hydrogels that can be manipulated to enhance

their efficacy as release vehicles,[12–15] biodegradability is a

remarkable strategy. There are several studies in the

literature reporting the controlled release of drugs from

biodegradable hydrogels,[16,17] such as the release of

recombinant human interleukin-2 from dextran-based

hydrogels;[18] similar dextran hydrogels were investigated

as drug delivery devices for the posterior part of the eye.[19]

Moreover, the polymer hyaluronic acid (HA) was used to

create a combination tissue engineering scaffold and protein

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D. Costa, A. J. M. Valente, M. G. Miguel, J. Queiroz

delivery vehicle.[20] Recently, in situ forming biodegradable

poly(ethylene glycol) based hydrogels were created for the

time-controlled release of tethered peptides or proteins[21]

and injectable biodegradable hydrogels were studied as

vehicles for the release of anticancer drugs.[22] Biodegradable

hydrogels appear to be quite suitable for DNA release, once

one can readily control the release rate by modulating the

network structure with adjusting crosslinking density.[23,24]

In an attempt to evolve in the cancer cure, a new strategy

seems to be the combination of chemical and gene therapy

improving the treatment efficacy due to their synergistic

effect. Simultaneously with anticancer drugs, specific genes

can be delivered to enhance sensitivity of targeted cells to

drug during all the treatment. Over the past decade, a few

studies reporting the delivery of nucleic acids and drugs

have been presented,[25–27] illustrating the advantage of its

combined effect in medical treatments. In this work, we

present a new p53 encoding plasmid DNA (pDNA) microgel

that is porous, biocompatible, photodegradable and thus,

suitable for the dual release of pDNA and anticancer

drugs and we add our contribution to the progress of curing

cancer through combined therapies. This bifunctionality

may represent an enormous advance in comparison to

individual chemotherapy treatments, and will certainly,

serve as a basis to improve cancer therapy.

2. Experimental Section

2.1. Materials

Ethylene glycol diglycidyl ether (EGDE) was from Aldrich. N,N,N0 ,N0

tetramethylethylenediamine (TEMED), sodium hydroxide (NaOH),

sodium bromide (NaBr), sorbitan monooleate (Span 80) and

poly(ethylene glycol)sorbitan monooleate (Tween 80), and doxor-

ubicin (DOX) hydrochloride were obtained from Sigma.

2.2. Plasmid Production and Purification

The 6.07 kbp plasmid pcDNA3-FLAG-p53 (Addgene plasmid

10838,Cambridge, MA, USA) used in the experiments was amplified

in a cell culture of Escherichia coli DH5a. Growth was carried out

at 37 8C in an Erlenmeyer flask with 500 mL of Terrific Broth

medium (20 g � L�1 of tryptone, 24 g � L�1 of yeast extract, 4 mL � L�1

of glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4) supplemented with

30 mg �mL�1 of ampicillin. Growth was suspended at late log phase

(OD600 nm�10). Cells were recovered by centrifugation and the

plasmid purified by the Qiagen Plasmid Maxi Kit (Qiagen, CA, USA)

according to the supplier’s protocol. Both pDNA isoforms, open

circular and supercoiled, are present.

2.3. Preparation of pDNA Microgels

pDNA microgels were prepared using an inverse microemulsion

polymerization method with a water-based gel solution dispersed

in an organic phase. The pDNA (2.0, 4.0, and 6.0 mg �mL�1) was

Macromol. Biosci. 2012, DOI: 1

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dissolved in aqueous buffer along with 0.1 or 0.2% EGDE, 10�3M

NaBr, 0.1 M NaOH, and TEMED. A 5 mL portion of this reaction

mixture was injected into 20 mL hexane containing 0.5 wt%

surfactants (Span 80/Tween 80) in a round-bottom flask under

heavy stirring. These neutral surfactants do not coordinate the

phosphate backbone of DNA, as would occur with ionic surfactants.

The polymerization continues at 40 8C for 20 min. The polymeriza-

tion and crosslinking take place simultaneously creating a gel

sphere from each solution droplet. Following the synthesis,

pDNA microgels were washed with excess hexane and dried

under vacuum, with yields ranging from 61 to 84%.

2.4. Scanning Electron Microscopy (SEM) Imaging

SEMwasused toevaluate the morphology ofplasmidDNA microgels.

Previously, the pDNA microgels were lyophilized overnight (�50 8C,

0.035 mbar). All the samples were kept under vacuum conditions and

taken out just before the SEM observation. The samples were

mounted on aluminum supports, fixed with double-sided adhesive

tape and sputter coated with gold using an Emitech K550 (London,

England) sputter coater. The samples were analyzed using a Hitachi

S-2700 (Tokyo, Japan) scanning electron microscope operated at an

accelerating voltage of 20 kV at various magnifications.

2.5. Determination of Encapsulation Efficiency

The plasmid DNA microgels were washed by dispersing them in

0.250 M sodium phosphate buffer at pH¼ 7.4 using a series of

sonication and vortex steps. The microgels were collected as a pellet

after centrifuging for 10 min and the supernatant was recovered.

The amount of non-bound pDNA was determined spectrophoto-

metrically measuring the absorbance at 260 nm using a Shimadzu

UV-Vis 1700 spectrophotometer. The encapsulation efficiency was

calculated according to

EEð%Þ ¼ ½ðTotal Amount of pDNA�Non-bound pDNAÞ=Total amount of pDNA� � 100

2.6. Cell Source and Growth

Normal human dermal fibroblast (NHDF) adult donor cells, Ref.

C-12302 (cryopreserved cells) from PromoCell were used. Cells were

plated in 175 cm3 T-flasks with Dulbecco’s modified Eagle medium

(DMEM) F12 (1:1 v/v) supplemented with heat-inactivated fetal

bovine serum (FBS, 10 vol% and L-glutamine (2� 10�3M). After 3 h, the

nonadherent cells were washed out. Cells were kept in culture at 37 8C,

in a humidified atmosphere, 5% CO2. After confluence was attained,

cells were subcultivated by a 5-min incubation in 0.20% trypsin

(1:250) and 5�10�3M ethylenediaminetetraacetic acid (EDTA).

Following centrifugation, cells were resuspended in sufficient culture

medium and seeded in 96-well plates containing the pDNA gels.

2.7. Determination of pDNA Gel Cytotoxicity by MTT

Assay

3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

(MTT) is a yellow, water-soluble tetrazolium salt. The MTT assay

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Table 1. Encapsulation efficiency of the crosslinked pDNA micro-gels.

EGDE

[%]

Initial pDNA

loading

[mg mL�1]

Encapsulation

efficiency [%]

pDNA DOX

0.1 2 42

4 44 79a)

6 45

0.2 2 42

4 42 72a)

6 44

a)Initial DOX loading amount is 5 wt%.

Plasmid DNA Microgels for a Therapeutical Strategy Combining the Delivery of Genes . . .

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is a simple non-radioactive colorimetric assay to measure cell

cytotoxicity or viability. Metabolically active cells are able to

convert this dye into a water-insoluble dark blue formazan by

reductive cleavage of the tetrazolium ring. The formed crystals can

be dissolved and quantified by measuring the absorbance of the

solution at 570 nm.

Plasmid DNA microgels were applied to a 96-well plate (Nunc.).

Before cell seeding, the plates were ultraviolet irradiated for 30 min.

Human fibroblast cells were plated at a density of 104 cells per

well in 96 well plate at 37 8C in 5% CO2 humidified atmosphere, for

24 and 48 h. After incubation, the redox activity was assessed

through the reduction of the MTT. 100 mL of MTT dye solution

(0.05 mg �mL�1 in Krebs) was added to each well, followed by

incubation for 2 h at 37 8C, in a 5% CO2 atmosphere. The medium

was aspirated and cells were treated with 50 mL of isopropyl

alcohol/HCl (0.04 N) for 30 min. The absorbance at 570 nm was

measured using a Biorad Microplate Reader Benchmark. The

spectrophotometer was calibrated to zero absorbance using

culture medium without cells. The relative cell viability (%) related

to control wells was calculated by [A]test/[A]control� 100, where

[A]test is the absorbance of the test sample and [A]control is the

absorbance of control sample. All the experiments were repeated

three times in triplicate. The statistical analysis of experimental

data used the Student’s t-test and the results were presented as

mean� standard deviation. Statistical significance was accepted

at a level of p< 0.05.

2.8. pDNA Gel Degradation by Light Irradiation

The pDNA microgels were irradiated with light (400 nm), in order to

promote the microgel photodisruption and consequent release of

pDNA.

2.9. pDNA Release Measurements

To determine the amount of pDNA released, the microgels were

incubated at 25 8C, in 20 mL of pH¼7.6 10�10�3 m Tris/HCl buffer.

At certain time intervals, the beads were collected by centrifuga-

tion and the supernatant analyzed. Plasmid DNA released into the

supernatant was quantified by spectrophotometrically measuring

the absorbance at 260 nm using a Shimadzu UV-Vis 1700 spectro-

photometer.

2.10. Incorporation and Release of DOX

DOX has been incorporated into pDNA gels by imbibition.

Concentrated solutions of the drug (5 wt%) were prepared. The

dried microgels were saturated with DOX by placing two micro-

spheres in 50 mL of each solute solution. The microgels were left

in the solutions for approximately 48 h. The DOX microgels

were collected as a pellet after centrifuging for 10 min and

the supernatant was recovered. The DOX concentration was

measured using Ultraviolet-Visible spectrophotometry at 497 nm.

The encapsulation efficiency was calculated according to

EEð%Þ ¼ ½ðTotal Amount of DOX

�Non-bound DOXÞ=Total amount of DOX� � 100

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Macromol. Biosci. 2012, DOI: 10.1



DOX loading efficiencies are reported in the Table 1. During DOX

loading, pDNA microgels kept their integrity and no pDNA has been

released.

In the release experiments, DOX loaded microgels were placed

in phosphate buffer saline (PBS, pH¼7.4); aliquots of the sample

solutions were withdrawn at appropriate time intervals and the

volume of the medium was kept constant by replacement. The DOX

concentration was measured spectrophotometrically. To avoid

interference of microgel degradation products on the measurement

of the drug concentration, the solutions from identical pDNA

microgels were used as a blank in measuring the DOX concentra-

tion as a function of degradation.

2.11. Release Models

The release mechanism has been assessed through the Korse-

meyer-Peppas equation[28]

002/m

H & Co

the

Ct=C1 ¼ ktn (1)

where Ct and C11 are cumulative concentrations of the material

released at time t and at infinite time, respectively, and k and n are

fitting parameters, giving the later useful information on the release

mechanism; from Equation 1, the mean dissolution time (MDT),

which characterizes the drug release rate from a dosage form and to

indicate the drug-release-retarding efficiency of the polymer[29] can

be calculated through the following equation[30]

MDT ¼ n

nþ 1

� �k�n�1

(2)

The application of Equation 1 is restricted to cumulative release

smaller than 60%. Further insight into the release mechanism is

obtained by fitting the amount of drug remaining in the matrix at

time t, Q, to the Hixson-Crowell cubic root equation[31]

Q1=3 ¼ Q1=30 � kCt (3)

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where Q0 is the amount of drug in the matrix at t¼ 0 and kC is the

cube root law rate constant. This equation describes the release

from systems where a change in surface area and diameter of

particles occurs. The release kinetics is evaluated through zero

order and first order rate law equations (Equation 4 and 5,

respectively); the former describes a system where the drug

release is independent of its concentrations, whilst for the latter

the release rate is concentration-dependent.

rly V

Q ¼ Q0 � k0t (4)

lnQ ¼ lnQ0 � k1t (5)

quation 4 and 5, k and k are zero- and first-order rate
In E 0 1
constants, respectively.

Both the release kinetics and the mechanism of release are

analyzed by using the Weibull function

Ct ¼ C1½1� expð�kWtÞd� (6)

where kW and d are constants related to the release rate and

mechanism, respectively.[32]

2.12. Cell Culture and in vitro Transfection

Cancer cells HeLa were incubated at 37 8C in a humidified

atmosphere containing 5% CO2 and maintained in DMEM

with high glucose (DMEM-HG) (Sigma) supplemented with

10% heat inactivated fetal calf serum, 0.5 g � L�1 sodium bicarbo-

nate, 1.10 g � L�1 4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid (HEPES), and 100 mg �mL�1 of streptomycin and 100 U �mL�1

of penicillin (Sigma). Cells were seeded in 25 cm3 T-flasks until

confluence was attained. Afterward the cells were sub-cultivated

by incubation on 0.18% trypsin (1:250) with 5� 10�3M EDTA.

Transfection studies were performed by seeding the cells in

24 well-plates at a density of 6�104 cells per well in 1 mL DMEM

and incubated for 24 h. The complete medium was replaced by

500 mL of medium supplemented with 10% FBS and without

antibiotic, in order to promote transfection; moreover, lipo-

fectamine, a transfection reagent, was used. Before being used

in transfection studies, the pDNA microgels were irradiated

with light (400 nm), in order to promote the microgel photo-

disruption and consequent release of pDNA. Thereafter, the

pDNA/lipofectamine complex was prepared as per the protocol

described by the supplier of the reagent (Invitrogen, Carlsbad, CA,

USA). In a typical procedure, a pDNA microgels and Lipofectamine

were mixed together and incubated at room temperature for

30 min to allow the complexation to take place. The mixture (75 mL)

was added to each well for transfection. The cells were then

incubated for a period of 6 h and then supplemented with DMEM.

After in vitro transfection (at least, n¼3) studies proceeded with

the quantification of p53 protein by Pan-Elisa analysis.

2.13. Determination of p53 Protein Content

For the detection of p53 protein, we used a p53 pan-Elisa kit (Roche

Applied Science). This assay for the quantification of wild-type and



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mutant p53 of human, mouse and rat origin is based on a

quantitative sandwich Elisa principle. The procedure starts with

sample preparation (detergent lysis of cells) followed by centrifu-

gation. Transfected cell lysates were prepared from detergent lysis

(lysis buffer-tissue, 50:50 v/v) in lysis buffer (1% Triton X-100 and

0.1% sodium dodecylsulfate (SDS) in PBS, pH¼7.4 and proteinase

inhibitor cocktail) and homogenization. The biotin-labeled capture

antibody is prebound to the streptavidin-coated microtiter plate.

During a single incubation step, the p53-containing sample reacts

with the capture antibody and the peroxidase-labeled detection

antibody to form a stable immunocomplex. Subsequent to the

washing step, the peroxidase bound in the complex is developed by

tetramethylbenzidine as substrate. Finally, we can determine the

amount of colored product, and thus the concentration of p53, by

spectrophotometrically measuring the absorbance at 450 nm using

a Shimadzu UV-Vis 1700 spectrophotometer. All the experiments

were repeated three times in triplicate. The statistical analysis of

experimental data used the Student’s t-test and the results were

presented as mean� standard deviation. Statistical significance

was accepted at a level of p<0.05.

3. Results and Discussion

3.1. Synthesis and Characterization of pDNA

Microgels

Plasmid DNA microgels were prepared using an inverse

microemulsion polymerization method with a water-based

gel solution dispersed in an organic phase. We produced

pDNA microgels (0.1 or 0.2% EGDE) with spherical

conformation and heterogeneous size distribution. The

diameter of pDNA-based microgels ranged from 0.1 to 2 mm,

and from 1 to 5 mm for microspheres crosslinked with 0.1

and 0.2% EGDE, respectively. Higher crosslinking density

used in the formulation synthesis leads to microgels with

larger diameters. Concerning the different pDNA loading

amounts for each crosslinker density used, similar mean

diameters were found illustrating that the EGDE density is

the most important aspect in controlling microgels size

distributions. Figure 1 presents the scanning electron

micrograph of pDNA microgels crosslinked with 0.1%

EGDE and 6 mg �mL�1 of pDNA loading. For each EGDE

density, microgels encapsulation efficiency depends on

the initial pDNA loading amount (Table 1). Increasing the

concentration of plasmid DNA in the polymerization

mixture from 2 to 4 and to 6, higher pDNA loadings

within the resulting microgels are obtained. Although, the

encapsulation efficiency remained fairly constant between

the two EGDE percentages, it is important to relate the

larger efficiency for pDNA microgels crosslinked with 0.1%

EGDE. Due to the higher encapsulation efficiency, our study

proceeded by using both 0.1 or 0.2% EGDE microgels and

6.0 mg �mL�1 pDNA. To determine the pDNA microgels

cytotoxicity, the MTT assay was performed. The MTT assay

is a simple non-radioactive colorimetric assay to measure

0.1002/mabi.201200096



Figure 1. Scanning electron micrograph of plasmid DNA microgels(0.1% crosslinker).


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cell cytotoxicity or viability. The MTT assays were

performed at 1 and 2 d after cells having been seeded on

top of 0.1 and 0.2% EGDE pDNA (6.0 mg �mL�1) microgels.

Our results, presented in Figure 2, suggest that both pDNA

microgels are non-toxic to cells, since every formulation

promoted dehydrogenase activity, with 0.1% EGDE gels

being less toxic. After 1 d, the fibroblasts were found to be 92

and 84% viable relative to control cells, for 0.1 and 0.2%

EGDE, respectively. After 2 d incubation with microgels, the

percentage cell viability slightly decreased (90 and 83% for

0.1 and 0.2% EGDE, respectively). Thus, the presented

formulation does not have an acute cytotoxic effect, and

thus, this system should not elicit an inflammatory

0

20

40

60

80

100

Cel

l Via

bilit

y (%

)

92% 90%

0.1% EGDE 0.2% EGDE1 Day 2 Days 1 Day 2 Days

84% 83%

Figure 2. Cytotoxicity profile of plasmid DNA microgels on humanfibroblasts after 1 and 2 d incubation as measured by MTT assay.Percent viability is expressed relative to control cells. The datawere obtained by calculating the average of three experiments.The respective errors were determined and were below 0.05%.





response that can ultimately result in failure to achieve

normal cell growth and function. Moreover, it was crucial to

demonstrate that the microgels actually protect the

encapsulated pDNA against serum nucleases, since this is

an important issue affecting both pDNA stability and

transfection efficiency. The pDNA microgels were exposed

to serum nucleases by combining them with DMEM

containing 10% FBS at 37 8C. If any degradation occurs by

the nucleases present in this medium, it would lead to

small and linear fragments, and this short pDNA sequences

would readily diffuse out from the microgels. Fortunately,

as seen in Figure 3, no appreciable decrease in the

concentration of double-stranded pDNA was observed

with the pDNA encapsulation over a period of 96 h. In

contrast, it was found that naked pDNA was completely

degraded after 1 h of incubation with serum nucleases

(Figure 3). This reinforces the fact that pDNA is really located

within the microgels; moreover, the pDNA integrity is

maintained which makes this system suitable as a pDNA

carrier for gene delivery applications in vivo.

3.2. Controlled Release of pDNA and DOX

Ethylene glycol ethers photo-oxidize in the presence of

the sun light.[33] The degradation on ultraviolet light

exposure (photo-oxidation) of EGDE leads to the removal

of the chemical crosslinks and can allow the release of

the constituent network polymer inducing changes in gel

weight, mechanical properties, mesh size, porosity, and

in the degree of swelling.[24] In order to demonstrate the

plasmid DNA microgel disruption, experiments on plasmid

DNA release were performed after the microspheres being

0

20

40

60

80

100

% D

NA

Rec

over

ed

0 8 12 24 48 96

Time exposed to DNase I (h)

Figure 3. Stability of 0.1% EGDE pDNA-encapsulated microgelsafter incubation with DNAse I. The data were obtained bycalculating the average of three experiments. The respectiveerrors were determined and were below 0.05%.

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irradiated with light (400 nm) and in the dark, for pDNA

microgels crosslinked with 0.1 and 0.2% EGDE, as illustrated

in Figure 4 (left). After irradiation, both gels suffered

disruption leading to the release of plasmid DNA with time.

For 0.1 and 0.2% EGDE crosslinked gels, pDNA release

behavior presents a narrow time lag in the first 8 and 12 h,

respectively, after which the release gradually increases

until a plateau is reached around 140 h of photodegrada-

tion. The initial time lag may be related to the number of

crosslinks that have to be degraded to permit the release of

plasmid DNA. After irradiation, and at maximum release,

pDNA gels crosslinked with 0.1% EGDE released 96.9% of

plasmid DNA while gels prepared from 0.2% EGDE released

88.8% of pDNA, in approximately 6 d. It is noted that the

extent of pDNA release is quite dependent on crosslinker

density, as found before for similar systems;[24] the higher

the crosslinker concentration used in the microgel pre-

paration, the lower the amount of plasmid DNA released.

Completely different behavior was found for release

studies performed in dark conditions for 0.2% EGDE pDNA

microgels (Figure 4). In the absence of light irradiation

minimal amounts of plasmid DNA are released, less

than 2%. For 0.1% EGDE pDNA microgels, the effect of

0 50 100 1500

20

40

60

80

100

Cum

ulat

ive

pDN

A R

elea

se (%

)

Time (h)

A-Photodegradation

B-Dark

0.1% EGDE

0 50 100 1500

20

40

60

80

100

Cum

ulat

ive

pDN

A R

elea

se (%

)

Time (h)

B-Dark

A-Photodegradation0.2% EGDE

Figure 4. Cumulative release of pDNA and DOX from pDNA microgels (Studies were performed after the irradiation of microgels with light (of experimental data to Equation 6.




photodisruption is less relevant since the pDNA release

profile is similar in the absence of light. In the dark

conditions, 77.9% of pDNA is released, in approximately

144 h. In this system, the use of light leads only to a

larger pDNA release percentage. In this sense, the induced

disruption can control the amount of pDNA released but

it cannot efficiently be used as a trigger to control the

release of pDNA. The interesting behavior displayed by

these microgels gives us the possibility to use both, the

photodisruption and crosslinker density, as significant

tools to modulate and optimize the pDNA release.[32]

Moreover, the several and singular properties reported can

be employed in a variety of specific and desired gene

delivery applications and this versatility appears as a

valuable asset.

In this study, DOX, one of the most common chemother-

apeutic drugs was chosen for its poor solubility and easy

detection by fluorescence monitors to demonstrate the

drug and gene co-delivery potential of pDNA microgels. The

anticancer drug was loaded into both pDNA microgels and

encapsulation efficiencies are presented in Table 1. High

encapsulation efficiencies are found, with a larger DOX

loading for the less crosslinked microgel. The cytotoxicity of

0 50 100 150 2000

20

40

60

80

100

Cum

ulat

ive

Dox

orub

icin

Rel

ease

(%)

Time (h)

A-Photodegradation

B-Dark

0.1% EGDE

0 50 100 150 2000

20

40

60

80

1000.2% EGDE

B-Dark

A-Photodegradation

Cum

ulat

ive

Dox

orub

icin

Rel

ease

(%)

Time (h)

0.1 and 0.2% w/v EGDE and 6 mg �mL�1 of pDNA) as a function of time.400 nm) (A) and in the dark (B). Solid lines correspond to best fitting

0.1002/mabi.201200096



0

20

40

60

80

100

Cel

l Via

bilit

y (%

)

Dox alone 0.1% pDNA-DOX 0.2% pDNA-DOX

2 Days

59%

73%

64%

Figure 5. Cytotoxicity profile of DOX and plasmid DNA-DOXmicrogels on human fibroblasts after 2 d incubation as measuredby MTT assay. Percent viability is expressed relative to controlcells. The data were obtained by calculating the average of threeexperiments. The respective errors were determined and werebelow 0.05%.


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the formed complexes was assessed by the MTT assay.

The results, available in Figure 5, indicate a decrease in

cytotoxicity for DOX loaded microgels, 73 and 64% cell

viability for 0.1 and 0.2% EGDE, respectively, when

compared with control samples of DOX alone, which

presents a cell viability of 59%. It appears that the

encapsulation of DOX into pDNA microgels reduces the

cell toxicity, in particular for the less crosslinked system,

Table 2. Fitting parameters of Equation 1–6 to experimental release

Equation Parameter EGDE 0.1%

Photo D

1a) n 1.76� 0.07) 2.07

R2 0.9880 0

MDT [h] 27

3 kC [h�1/3] (6.5� 0.1)� 10�3 (3.8� 0

R2 0.9928 0

4 k0 [h�1] (1.05� 0.06)� 10�2 (7.8� 0

R2 0.8884 0

5 k1 [h�1] (2.71� 0.08)� 10�2 (1.33� 0

R2 0.9746 0

6 kW [h�1] 2.53 (�0.09)� 10�2 1.91 (�0

d 1.59 (�0.08) 1.9

R2 0.9949 0

a)Fitting has been performed to cumulative release <60%.





enhancing its biological use. In vitro release profile of

DOX from 0.1 to 0.2% EGDE pDNA microgels is shown in

Figure 4 (right side). As discussed above for pDNA release,

the irradiation of 0.1% EGDE pDNA microgels with light has

a minor effect in the DOX release; similar release behavior

was found in the dark conditions, however, with low drug

released amount. In both cases, the DOX release exhibits

two-phase drug release, where burst effect occurs at the

initial 8 h, and then gradually increases until a plateau is

reached around 200 h. At this time, 97.8 and 86.9% of DOX

is released from 0.1% EGDE pDNA microgels in the presence

and absence of light, respectively. Using the strategy of

network disruption one can increase, in approximately

10%, the drug released amount. Different situation was

observed for 0.2% EGDE pDNA microgels, where the effect

of photodisruption is quite pronounced; the DOX release

in the absence of light is close to 4.5%. Induced pDNA

microgel disruption promotes the drug release with a trend

presenting, similarly to 0.1% EGDE microgels, a small burst

in the first 8 h. After 72 h of photodisruption, the DOX

release comes to a slow level and after around 8 d 93.6%

of DOX is released.

The mechanism and kinetics of pDNA and DOX

release from pDNA gel matrices were evaluated by fitting

the experimental release data (Figure 4) to a set of

appropriate equations, available in Section 2. Table 2

and 3 show fitting parameters calculated by fitting the

experimental cumulative release data from Figure 4, for the

simultaneous transport of pDNA and DOX from pDNA-DOX

gel matrices, to Equation 1–6 presented in Section 2. pDNA

release follows a Super-Case II mechanism, characterized by

n values higher than 0.89 (for cylindrical shape matrices)

data of pDNA from pDNA-DOX gel matrices to water, at 25 8C.

EGDE 0.2%

ark Photo Dark

� 0.07 2.02� 0.09 1.7� 0.1

.9899 0.9860 0.9731

37 26 24

.1)� 10�3 (5.8� 0.2)� 10�3 (1.17� 0.07)� 10�4

.9793 0.9808 0.9548

.3)� 10�3 (9.9� 0.6)� 10�3 (2.3� 0.2)� 10�4

.9506 0.8511 0.7521

.05)� 10�2 (2.12� 0.08)� 10�2 (2.3� 0.2)� 10�4

.9591 0.9559 0.7544

.07)� 10�2 2.91 (�0.08)� 10�2 3.5 (�0.2)� 10�4

(�0.1) 2.0 (�0.1) 1.7 (�0.2)

.9941 0.9955 0.9846

002/mabi.201200096



Table 3. Fitting parameters of Equation 1–6) to experimental release data of DOX previously loaded in pDNA gels to water, at 25 8C.

Equation Parameter EGDE 0.1% EGDE 0.2%

Photo Dark Photo Dark

1a) n 0.33� 0.02 0.61� 0.08 0.44� 0.04 0.8� 0.2b)

R2 0.9876 0.9479 0.9726 0.9230

MDT [h] 52 48 44 17

3 kC [h�1/3] (5.6� 0.3)� 10�3 (3.9� 0.1)� 10�3 (4.8� 0.2)� 10�3 (1.5� 0.2)� 10�4

R2 0.9777 0.9916 0.9813 0.9014

4 k0 [h�1] (6.7� 0.7)� 10�3 (5.8� 0.4)� 10�3 (6.4� 0.6)� 10�3 (3.1� 0.3)� 10�4

R2 0.5581 0.8034 0.86048 0.6127

5 k1 [h�1] (2.25� 0.07)� 10�2 (1.25� 0.05)� 10�2 (1.66� 0.06)� 10�2 (3.1� 0.3)� 10�4

R2 0.9896 0.9770 0.9823 0.6196

6 kW [h�1] (2.4� 0.09)� 10�2 (1.4� 0.03)� 10�2 (1.9� 0.06)� 10�2 (3.19� 0.03)� 10�4

d 0.6� 0.1 fickian 0.9� 0.1 0.7� 0.1 0.99� 0.09

R2 0.9648 0.9870 0.9837 0.9911

a)Fitting has been performed to cumulative release <60%; b)A time lag of 2.9 h has been considered.

8

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www.mbs-journal.de


independently on crosslinker concentration or presence/

absence of light.[24] This mechanism emerges as the

polymer resistance becomes more significant relative to

the diffusion resistance; consequently, the release is linear

at early times in the process, but at long time the release

rate increases markedly; it can also be expected that the

release of pDNA will change, by dissolution, the surface area

and gel matrix dimensions; in fact, a good fitting to the

Hixson-Crowell cubic root equation (Equation 3) is obtained

confirming that assumption. Looking to the MDT para-

meter, which characterizes the drug release rate from a

dosage form and indicates the drug-release-retarding

efficiency of the polymer, the pDNA MDT increases by

increasing the crosslinker concentration (from 24 to 36 h)

in the absence of light but it remains approximately

constant when the release takes place in photo conditions.

In general, the pDNA release (or dissolution) is characterized

by a first-order kinetic law, with rate constants decreasing

by increasing the crosslinker concentration; it is also

worth noting that the use of light not only leads to a higher

percentage of pDNA released but also to a higher release

rate. It should be stated that the poor fitting obtained for

the gel containing 0.2% EGDE (in dark conditions) is

probably due to the small (almost neglected) maximum

percentage of released pDNA. The equation that best fit

the entire set of pDNA release data is the Weibull function

(Equation 6). This empirical equation gives, simultaneously,

information on the mechanism (d) and rate kW of release.

d values are higher than 1, indicating that a complex

mechanism, which corresponds to a Super-Case II transport,

governs the release process;[34] concomitantly, the rate




constants (kW) are quite similar to those calculated through

a first order rate law equation. Consequently, the Weibull

function seems to be reliable for an assessment of both:

mechanism (d) and rate kW of release of pDNA. The DOX

release shows different features when compared with the

pDNA release profile. The drug release mechanism is Fickian

(n¼ 0.44) (or pseudo-Fickian for 0.1% EDGE pDNA, n¼ 0.33)

when the gel matrices are irradiated by light, and shows an

anomalous behavior (0.45<n< 0.89) at dark conditions.[35]

A Fickian release is characterized by a diffusion-controlled

transport, whilst an anomalous transport occurs due to a

coupling of Fickian diffusion and polymer relaxation. The

drug MDT follows the same trend as that found for pDNA;

however, for 0.1% EGDE pDNA gels, in the presence and

absence of light, MDT are equal to 52 and 48 h, respectively;

these values are two times higher than the normal

required duration of release, i.e., 24 h. The kinetics of

DOX release follows a first-order law equation (with

exception of 0.2% EGDE pDNA in dark conditions, where

a scarce number of points, due to the maximum DOX release

being only 4%, does not allow any conclusion). Although

mechanisms of simultaneous transport of pDNA and

DOX are significantly different, rate constants are similar;

furthermore, these rate constants values (k1) are in good

agreement with those obtained by the Weibull

equation (kW). From the previous discussion and taking

into account the effect of pDNA disruption on DOX release,

confirmed by a reasonable fitting of DOX experimental data

to the Hixson-Crowell cubic root equation, it can be

hypothesized that the drug diffuses out through an outer

layer which erodes allowing the aqueous medium to

0.1002/mabi.201200096



2500

3000

rote

in)

DOX

0.1 % EGDE


www.mbs-journal.de

penetrate further into the core;[36] this effect seems to be

more significant in the absence of light than in its presence

for pDNA-based gels.

0

500

1000

1500

2000

Rel

ativ

e p5

3 D

ensi

ty (U

nits

/mg

P

DOX

4 6 6 4 6 6 DNA Loading (µg/ml)

0.2% EGDE

Figure 6. Quantification of p53 protein expression in cancer HeLacells using a pan-p53 Elisa kit, for 0.1 and 0.2% w/v EGDEcrosslinked pDNA microgels (4 or 6 mg �mL�1 pDNA loading)complexed with lipofectamine. The data were obtained bycalculating the average of three independent experiments. Therespective errors were determined and were below 0.05%.

3.3. In vitro Transfection

For the cellular internalization of pDNA microgels, size is a

critical parameter and can compromise the process of gene

expression in targeted cells. The mean diameter of 0.1

and 0.2% EGDE pDNA microgels is between 0.1 and 2 mm,

and 1 and 5 mm, respectively. Due to the smaller size,

we hypothesize that the less crosslinked system would

have better transfection capacity. It is known, from the

literature, that particles with similar size are suitable

for the intracellular delivery of macromolecules and to

promote cellular transfection leading to encoded protein

expression.[37,38] The tumor-suppressor gene p53 encodes

a phosphoprotein whose expression and function are

involved in malignancy, apoptosis, and other abnormal

cell proliferation processes. Before being used in transfec-

tion studies, the pDNA microgels were irradiated with light

(400 nm), in order to promote the microgel photodisruption

and consequent release of pDNA. The p53 protein content

has been determined by using the p53 pan-Elisa kit. This

assay for the quantification of wild-type and mutant p53 is

based on a quantitative sandwich Elisa principle. Figure 6

presents the relative quantification of p53 protein expres-

sion in cancer HeLa cells transfected with 0.1 and 0.2% EGDE

pDNA microgels, as a function of initial pDNA loading

amount. From the obtained data, we can state that 0.1%

EGDE microgels are able to surpass the cellular barrier and

release bioactive pDNA into the cytosol. The barrier of the

nucleus is permeable to foreign pDNA only during cell

division; the high rate of tumor cell division, probably,

enhances transfection and gene expression. Additionally,

we verify that a larger amount of pDNA loading, in

0.1% EGDE microgels, increases the transfection efficiency

leading to higher levels of protein expression. Contrary, for

the most crosslinked pDNA vector the success of transfec-

tion is very limited since the quantified p53 density is

not significant. Moreover, the effect of increasing pDNA

loading, only slightly enhances the p53 protein content.

For this situation, contributes the mean diameter of 0.2%

pDNA microgels which is too large for efficient intracellular

uptake and internalization. It becomes clear the relevance

of vector properties such as, size, crosslinker density, and

pDNA loading in the success of transfection process, of

which, size seems to be the most important of all. Thus, the

less crosslinked microgel carrier is more suitable for gene

delivery applications since it demonstrates a good biolo-

gical functionality for pDNA loading and in vitro gene

expression. Finally, in both systems, the incorporation of

DOX in the microgels seems do not interfere in the

transfection capacity neither affect the relative p53 density,





as evidenced in Figure 6, for the highest pDNA loading 0.1

and 0.2% EGDE microgels.

4. Conclusion

We have succeeded in preparing a new plasmid DNA

microgel by an inverse microemulsion polymerization

method. These microgels have a suitable size, are biocom-

patible and able to protect DNA against DNAse degradation.

Furthermore, they can load larger quantities of pDNA

and anticancer drugs and release them in a sustained and

controlled manner. Through a quantitative analysis using

appropriate release models we found that the pDNA release

follows a Super-Case II transport mechanism while the DOX

is Fickian when the microgels were light irradiated. For the

success of cellular transfection and consequent protein p53

expression, size is a critical parameter. The pDNA microgels

are good candidates for potential anticancer drug and

gene co-delivery system to achieve the synergistic effect

of chemical and gene therapies improving the success of

medical cancer treatment. In order to improve transfection

and expression rate in eukaryotic cells, and taking into

account the guidelines from regulatory agencies, our

research will proceed with the supercoiled (sc) isoform

purification of a p53 encoding plasmid. Moreover, in vivo

research may be followed to state the efficacy of this Pdna-

based carrier for the application of animal experiment.

Acknowledgements: We are grateful for financial support fromFundacao para a Ciencia e a Tecnologia (FCT), (SFRH/BPD/47229/2008) and to PTDC/EBB-Bio/114320/2009. The authors would like

002/mabi.201200096



10

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to thank to Fani Sousa e Angela Sousa for the 6.07 kbp plasmidpcDNA3-FLAG-p53 production and recovery.

Received: March 19, 2012; Revised: May 10, 2012; Publishedonline: DOI: 10.1002/mabi.201200096

Keywords: biocompatibility; co-delivery; in vitro transfection;microgels; pDNA delivery

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plasmid dna microgels for a therapeutical strategy combining the delivery of genes and anticancer...

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