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Design of polyamines-grafted starches for nucleotide analogues delivery: in vitro evaluation of the anticancer activity Erdem Kanber, Hiroe Yamada, Brigitta Loretz, Elise Lepeltier, and Claus-Michael Lehr Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.6b00396 • Publication Date (Web): 15 Sep 2016 Downloaded from http://pubs.acs.org on September 17, 2016

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Bioconjugate Chemistry

Design of polyamines-grafted starches for nucleotide analogues delivery: in vitro evaluation of the anticancer activity Erdem Kanber1, Hiroe Yamada1, Loretz Brigitta1, Elise Lepeltier 2*, Claus-Michael Lehr1,3 1

Drug Delivery (DDEL), Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),

Helmholtz Centre for Infection Research (HZI), 66123 Saarbrücken, Germany 2

INSERM U1066 Micro & Nanomed Biomimetique, 4 rue Larrey, 49933 Angers, France

3

Department of Pharmacy, Saarland University, 66123 Saarbrücken, Germany

*E-mail: [email protected] Abstract Nucleotide analogues are a therapeutic class really promising and currently used in clinic notably against viral infectious diseases and cancer. However, their therapeutic potential is often restricted by a poor stability in vivo, the induction of severe side effects and a limited passive intracellular diffusion due to their hydrophilicity. Polysaccharide-based polymers (e. g. starch) have considerable advantages including a lack of toxicity and absence of antigenicity. The aim of this study was to develop new cationic starches able to form complexes with nucleotide analogues: thus protect them and increase their cell uptake. At the same time, the material should demonstrate good biocompatibility and low cytotoxicity. Different polyamines, (TREN, TEPA and spermine) were grafted to starch in order to evaluate the impact of side chain properties. The resulting cationic starch derivatives were characterized (e.g. degree of modification) and compared in their properties to form polyplexes with ATP as a model nucleotide. Among the 1 ACS Paragon Plus Environment


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tested candidates, the formulation of starch-TEPA and ATP with a N/P ratio = 2 led to nanoparticles with a size of 429 nm, a PdI of 0.054 and a zeta potential of -9 mV. MTT and LDH assays on A549 cell line showed a low toxicity of this polymer. Confocal microscopy study proved that the cell internalization was an incubation time and energy dependent process. Most important, starch-TEPA complexed with ddGTP showed a significant biological activity on A549 cancer cells compared to the plain ddGTP at the same concentration. Introduction Nucleoside and nucleotide analogues (NAs) are well-known for their antiviral and anticancer activity.1 They are synthetic, chemically modified compounds that have been developed to mimic their physiological counterparts. Antiviral NAs inhibit cellular division and viral replication whereas anticancer NAs inhibit cellular DNA replication and repair.2 NAs are generally administered in non-phosphorylated or more soluble 5'-monophosphorylated therapeutic form because nucleotide analogues 5'-triphosphates are usually considered too unstable as a drug form to be used directly.3 Despite its capability to reduce cancerous cells proliferation, NAs present some disadvantages, such as phenomenon of resistance, low oral bioavailability and poor in vivo stability due to its rapid degradation by serum nucleases when injected intravenously.4 A limited passive intracellular diffusion and further side effects are also observed, partially caused by long-term toxicity due to the hydrophilicity of the molecule and an ineffective intracellular conversion of these molecules into 5'-triphosphate.5 To use a nanocarrier in order to protect the active substance (from hydrolysis), to facilitate its cellular uptake and to reduce resistance phenomena is a strategy of interest. Several kinds of nanoparticles are already well studied such as polymeric nanocarriers, liposomes or self-assemblies.6 For example, the spontaneous formation of nanoparticles has been observed when the Gemcitabine, Dideoxycytidine, Deoxycitidine, Dideoxyinosine, or AZT were covalently coupled with a 2 ACS Paragon Plus Environment

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molecule of squalene (SQ), precursor of cholesterol.7,8 The Gemcitabine-SQ nanoparticles even demonstrated a superior in vivo anticancer activity on P388 and L1210 subcutaneous tumors grafted on mice compared, to the free Gemcitabine.9,10 This strategy is really promising but the major drawback is the synthesis and purification of these different amphiphilic molecules in several steps leading to weak yields. The use of biocompatible polymers to complex NAs represents an interesting alternative. Those polyplexes result from the spontaneous electrostatic interaction between a cationic polymer and an anionic molecule such as nucleic acids or nucleotide analogues 5'-phosphates.11 It is an easy, fast and efficient way to prepare nanoparticles, in just mixing the two compounds in aqueous solution resulting, at optimal ratio, in a complexation efficacy of 100 % and a high drug loading. Several physicochemical parameters have to be considered because of their impact on the transfection efficiency and on the cytotoxicity: polymer molecular weight, degree of branching, cationic charge density, particle size, zeta potential and buffer capacity. In the literature, few articles deal with the vectorization of NAs by polymers. Vinogradov et al. demonstrated that a cross-linked network of branched PEI and poly(ethylene glycol) (PEG) molecules ("Nanogel") was able to form spontaneously polyplexes with the 5'-triphosphated form of the cytotoxic 5fluoroadenine arabinoside (fludarabine): a high drug loading (30 %) and a size range between 100-300 nm were obtained.3 They showed the efficacy of these polyplexes on A549 cell lines compared to the free-drug, due partly to a slow release from the polymeric network. The main problem remained the cytotoxicity of the carrier. That is why it is necessary to find other systems that will be able to be both efficient for transfection and less toxic. Polysaccharide-based polymers such as chitosan, starch or hyaluronic acid have considerable advantages as gene delivery vector in particular their excellent biocompatibility, low toxicity and immunogenicity.12 Among these carbohydrate polymers, starch has the additional advantages of 3 ACS Paragon Plus Environment


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a biodegradability not only by hydrolysis, but also by human enzymes, particularly αamylase.13,14 Recently, starch-graft-PEI polymers have been studied by Yamada et al. and they showed that starch-b-PEI was a good strategy to form polyplexes due to cationic charges enabling the complexation with anionic DNA, an excellent transfection efficacy through the cell membrane and an efficient endosomal escape.15 They showed that starting from 30 wt% of modification, all polymers were able to form polyplexes with relatively uniform size distributions and with mean sizes of 70-100 nm. Although they demonstrated a lower cytotoxicity of the synthesized starch-b-PEI polymers than b-PEI alone, this point has however to be definitively improved. Thus, in this study three oligoamines were covalently coupled to starch: spermine, tris(2-aminoethyl)amine (TREN) and tetraethylenepentamine (TEPA). These different cationic side chains have been already grafted on different backbones, as disulfideamide polymer16 or β-cyclodextrine17 and showed good transfection efficiency and low cytotoxicity. Thereby, the aim of this study was to develop new optimized cationic starches able to form polyplexes with triphosphate nucleotide analogues (ddATP: 2',3'-Dideoxyadenosine-5'Triphosphate, ddCTP: 2',3'-Dideoxycytidine-5'-Triphosphate and ddGTP: 2',3'Dideoxyguanosine-5'-Triphosphate have been tested) and to demonstrate interesting in vitro properties.

Results and Discussion Synthesis of starch-TEPA, starch-TREN and starch-spermine

Starch-TEPA, starch-TREN and starch-spermine (Scheme 1) were synthesized according to the same protocole described by Yamada et. al.15 4 ACS Paragon Plus Environment

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a

c

H N

H2N

H2N

NH2

NH2 N

N H

b

H N

H N

H2N

N H

NH2

NH2

Scheme 1. Molecular formula of spermine (a) TEPA (b) and TREN (c).

Briefly, the water-soluble oxidized starches were synthesized by TEMPO-mediated system.18,19 Oxidized starch was further separated into three fractions depending on its MW (“small”, “medium” and “large”) using Ultrafiltration system. Considering that previous results showed the superior potential of the largest MW starch backbone fraction,15 only “large” MW (> 100 kDa) of the oxidized starch backbones were used in this study. Then, a covalent coupling of TEPA, TREN or spermine via a DMTMM-mediated amidation was done20 : Scheme 2. OH O

R HO

HO

O

OH O

R

R

oxidation

n

TEMPO/NaOCl

HO

HO

COONa O

O HO

pH = 8.5

H2N

NH NH

Spermine DMTMM reagent

NH2

HO

HO

O

R

NH

OH O

R

HO

NH O

NH2

NH

O

O HO

HO

O

R

Scheme 2. Covalent coupling of spermine with starch after an oxidation.

After each synthesis, Gel Permeation Chromatography (GPC) experiments have been performed: the main objective was to analyze the molecular weights of the different cationic polymers obtained. Indeed, a second population of compounds could be formed, via a double conjugation 5 ACS Paragon Plus Environment


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of the oligoamine to the starch backbone. However, according to the different chromatograms (Figure S1), one population in terms of MW has been obtained, demonstrating the monoconjugation. Fourier Transform Infrared spectroscopy (FTIR) was carried out to confirm conjugation with the different polyamine chains (Figure 1). All the FTIR spectra highlighted the presence of the characteristic peak at 1000-1100 cm-1 arising from the C-O-C vibration of the sugar.21 For the oxidized starch spectrum, there is the typical peak at 1600 cm-1 of the C=O stretching from the carboxyl anion COONa. Further, this peak partly disappeared after coupling with the polyamine chain, and is replaced by two peaks at 1660 cm-1 and at 1550 cm-1 from the amide bond: the amide I peak (between 1600 and 1700 cm-1) is mainly associated with the C=O stretching vibration and the amide II results from the N-H bending vibration and from the C-N stretching vibration. The average yield of these three syntheses was of 70 %.

Figure 1. FTIR spectra of starch, oxidized starch and starch-spermine: the black square shows the successful coupling of the spermine.

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1

H NMR spectra of oxidized starch (Figure S2) showed δH values between 5.12 – 5.58 ppm that

were assigned to the anomeric 1H atom. The signals between 3.3 – 4.2 ppm were ascribed to the ring protons 2H, 3H, 4H, 5H.18,22 The degree of oxidation (DSCOONa) was determined by a colorimetric assay, according to Blumenkrantz et al..23 For the coupling reaction, two different oxidized starches were used: for the starch-spermine, DSCOONa = 29 ± 5 % and for the starchTREN and for starch-TEPA, DSCOONa = 55.6 ± 5 %. The degree of coupling was determined by 1H NMR in D2O. The protons from the oligoamine chains (spermine NH2-CH2-CH2- CH2: δ =1.4, 1.55, 2.6 ppm ; TEPA and TREN: NH2-CH2CH2, 2.29 – 3.46 ppm) could be easily observed on the 1H NMR spectra (Figure S2) and the different percentages of modification were 22.0 wt%, 32.0 wt% and 28.0 wt% for the starchspermine, starch-TEPA and starch-TREN respectively (Table 1).

Starch-

Starch-

Starch-

spermine

TEPA

TREN

DSCOONa

29 ± 5 %

55.6 ± 5%

55.6 ± 5%

Wt%

22.0 wt%

32.0 wt%

28.0 wt%

Table 1. Characteristic parameters of the different modified starch synthesized: percentage of oxidation (DSCOONa) and percentage of oligoamines grafted (wt%).

Polyplexes preparation and characterization

In order to form spontaneously polyplexes, aqueous solutions of modified starches and NAs were mixed: complexation should occur due to electrostatic interactions between cationic charges from modified starches and negative charges from NAs. In milliQ® water, ATP has four



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negative charges (pka = 6.5 and 1.5 for the three other phosphates24), TREN has three positive charges (pka > 8.425), TEPA three as well (pKa = 9.7, 9.5, 8.3, 5.0, 3.326) and spermine four (pka > 7.927). The influence of several important parameters on the properties of polyplexes was studied: the N/P ratio, the nature of the polyamine chain, the concentration of cationic starch and the nature of the NAs.

Influence of the N/P ratio and of the modified starch

To study the influence of the N/P ratio, ATP was used as a model. The results are summarized in the Table 2. Zeta Compounds

N/P ratio

Size [nm]

PdI

Potential [mV]

1

461.7 ± 2.6

0.093

-10.5 ± 0.2

2

429.0 ± 5.5

0.054

-9.0 ± 0.3

5

60.6 ± 8.6

1.000

18.3 ± 0.5

1

2337 ± 211.4

0.168

-4.6 ± 0.1

2

1907 ± 95.0

0.057

-3.4 ± 0.2

5

385* ± 23.8

0.357

30.6 ± 0.8

Starch-

1

247.0* ± 67.0

0.344

-27.5 ± 11.2

spermine/ATP

2

325.0* ± 21.0

0.424

-7.87 ± 0.7

5

423.4* ± 38.1

0.367

-7.11 ± 1.1

StarchTEPA/ATP

StarchTREN/ATP

Table 2. Measurements of particle size, polydispersity index (PdI) and zeta potential according to N/P ratio. Polyplexes suspensions are in water, analysis was performed 30 min after preparation. (*several populations were observed: the size written corresponds to the major population). Boldface indicates the selected sample used for further studies.


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With starch-spermine polyplexes, none N/P ratio led to monodispersed polyplexes: PdI > 0.3. In every case, a N/P ratio = 5 seemed to disfavor the formation of polyplexes. Indeed, at this ratio, the PdI obtained was of 1 for the starch-TEPA polyplexes and the complexation with starchTREN showed 3 populations of particles, notably aggregates with a size > 1000 nm. The zeta potential was a good indication of the polyplexes formation. Indeed, for the starch-TEPA and starch-TREN complexes, a negative zeta potential went with low PdI, presumably because a part of complexed ATP was on the particle surface. For the starch-TEPA and starch-TREN polyplexes, a N/P = 1 led to a lower zeta potential and larger polyplexes than with a N/P ratio = 2. As highlighted in bold (Table 2), the best candidate polymer to complex ATP was the starchTEPA. At N/P = 2, starch-TEPA/ATP polyplexes showed a size of 429 ± 5.5 nm, the lowest PdI of 0.054 and a zeta potential of -9.04 ± 0.27 mV. To explain this result, the percentage of modification of the starch backbone had probably an importance. An increase in the degree of modification (wt%) caused a decrease in polyplexe size and less polydispersity. The number of available positively charged amines seemed to be determining. Indeed, based on an independent binding model, T.-M. Ketola et al. showed that for both linear and branched PEI, the mechanism of polyplexes formation varies as a function of amine concentration.28 In our case, calculations revealed that the number of available cationic amines for starch-TEPA, starch-TREN and starch-spermine were respectively 3.1.1018, 2.3.1018 and 2.0.1018 per mg of modified starch. Since polyplexes with suitable size and PdI were formed only with starchTEPA, this compound was used for the rest of the study.

Compared with the complexation of DNA, the N/P ratio had to be low with NAs in order to obtain monodispersed polyplexes. The same observation was made by Vinogradov et al. where they observed polyplexes formation of cross-linked network of branched PEI and poly(ethylene glycol) (PEG) molecules with ATP at a N/P ratio of 33. 9 ACS Paragon Plus Environment


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The independent binding model, in contrast to the cooperative binding model, does not take into account the simultaneous or subsequent binding of other amine ligands at unoccupied phosphate sites. A study28 explored the complexation of DNA with polyethylenimine and poly(L-lysine) by a spectroscopic method. Applying the cooperative binding model for multivalent ligand binding to multisubunit substrate showed that at pH 7.4 the mechanism of these DNA-polyplexes formation changes from independent binding to cooperative binding at N/P close to 0.6. The change from negative to positive zeta potential took place close to a N/P ratio of 2. Large particle size were first obtained probably due to aggregation of polyplexes when they are at nearly a charge-neutral state29. At this point, all the DNA phosphate groups were bound by amine groups of the polymer and the polyplexe core had been formed. However, at higher N/P ratios, excess polymer bound to the nanoparticle core, forming a protective shell around it, and the particle size decreased: core-shell model.30 In our case, at N/P ratios > 5, several populations with high PdI were obtained: the excess of cationic polymer may destabilize the polyplexes. Considering starch/TEPA polyplexes, a N/P = 1 corresponds to nearly twice as much as negatively charges than positively charges and a N/P = 2 corresponds to a slight excess of positive charges which was optimum in our case to obtain the smallest monodispersed polyplexes.

Influence of the concentration of starch-TEPA

In a general manner, the concentration of the material in solution is another important parameter to control the size of nanoparticles. Indeed, in increasing the concentration of polymer, the size of particles could increase but the number of particles as well. That is why, the size of the polyplexes formed with ATP was studied according to three different concentrations of starch-TEPA, at a same N/P ratio of 2: Table 3.


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Concentration of

Size [nm]

PdI

1 mg/mL

429.0 ± 5.5

0.054

2 mg/mL

920.4 ± 14.2

0.176

5 mg/mL

1451.3 ± 282.1

0.255

Starch-TEPA

Table 3. Size and PdI of polyplexes composed of Starch-TEPA/ATP at N/P ratio = 2 according to the polymer concentration.

The results showed that when the concentration was multiplied by a factor 2 or 5, the sizes of the polyplexes were multiplied by a factor 2.1 and 3.4. In our case the results suggested that the particles size was really affected by starch-TEPA concentration. So, if the concentration in polymer became higher, an increase in particles size was probably preferred rather than an increase in particles number. The optimized concentration of 1mg/mL was used to minimize particle size, which is known to have an influence on the cell uptake.31

Stability and shape of starch-TEPA/ATP polyplexes

The stability of starch-TEPA/ATP polyplexes, prepared with N/P = 2, stored at 4 °C in water was followed by DLS (Figure S3). The particles were stable during 11 days, meaning that only one population was observed. However, the size increased from 429 ± 5.5 nm at day = 0 to 603 ± 3.2 nm at day = 11: an increase of 41 %. After 11 days, a second larger population appeared, meaning an aggregation of polyplexes. The relatively low zeta potential of these polyplexes (around -9 mV) could explain the weak stability in water: a zeta potential > +25 mV or < -25 mV



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leads to high degree of stability due to electrostatic repulsion.32 In further developments, a way to stabilize these particles (for example using a surfactant like PVA) should be found. Representative SEM pictures were taken of starch-TEPA/ATP polyplexes (Figure 2): a spherical shape could be clearly observed.

200 nm

200 nm

Figure 2. Representative SEM pictures of starch-TEPA/ATP particles: a spherical shape was clearly observed.

Complexation efficiency of starch-TEPA/ATP polyplexes

To determine the complexation efficiency (CE) of ATP with the starch-TEPA, a standard ATP calibration was done thanks to the ViaLight™ plus kit as described in Materials and Methods. After 3 washings of the polyplexes, the intensity of luminescence of the different supernatants was determined. Knowing the corresponding amount of ATP in the supernatant and the initial amount of ATP introduced in the formulation, the CE was calculated. A mean value of CE = 95.0 ± 2.4 % was obtained for three different experiments. The drug loading was of 47.5 ± 1.9 %.


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Influence of the NAs

ATP was first used as a model to optimize important formulation parameters in order to obtain small and homogenous particles. Different nucleotides analogues were then studied with starchTEPA, at a concentration of 1 mg/mL and a N/P ratio of 2 (Table 4): ddATP, ddGTP and ddCTP. Size [nm]

PdI

ATP

429.0 ± 5.5

0.054

ddATP

625.0 ± 48.3

0.929

ddGTP

633.0 ± 6.8

0.293

ddCTP

587.6 ± 51.7

1

Table 4. Size and PdI of polyplexes composed of Starch-TEPA at N/P ratio = 2, at 1 mg/mL according to the NA used.

All these formulations lead to larger and more polydispersed particles, even when the difference was “only” of two OH groups on the molecule: for ddATP, the PdI of polyplexes is of 0.93. It means that both OH groups played a significant role in the complexation, probably thanks to non-ionic bonds. In S.-T. Chou et al. they showed that hydrogen bonds between histidine richpeptide and phosphate from siRNA enhanced the stability of polyplexes.33 The OH from ATP could be hydrogen bond donor or acceptor and the amines from cationic starches as well: this non-ionic interaction may explain the difference of complexation behavior between ATP and ddATP with starch-TEPA. The complexation with ddCTP led to a PdI = 1: no polyplexe was observed. However, the starchTEPA/ddGTP polyplexes led to interesting polyplexes: a PdI of 0.293 and a size of 633.0 ± 6.8 13 ACS Paragon Plus Environment


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nm were observed. When we compare the molecular formula of these NAs, they have the same number of negative charges, but not the same number of chemical functions being able to form hydrogen bonds with starch-TEPA: the guanine group has 6 possible groups, the adenine 5 and the cytosine 4. So these non-ionic interactions stabilized probably the complexation between the base of NAs and the cationic starch.

Evaluation of the modified starch/NAs polyplexes as drug carrier Biocompatibility

a

b

Figure 3. MTT (a) and LDH (b) assays on A549 cell line with different concentrations of starchspermine (SPM), starch-TREN (TREN), starch-TEPA (TEPA) and oxidized starch (starchCOO): HBSS buffer in cell medium was considered as the 100 % of cell viability and the Triton X-100 (TrX) solution as the 100 % of cytotoxicity.

The biocompatibility of the different polymers synthesized was tested on A459 cells, adenocarcinomic human alveolar basal epithelial cells: Figure 3. LDH is a soluble cytoplasmic enzyme that is present in almost all cells and is released into the extracellular space when the plasma membrane is damaged. According to this assay (Figure 3b), the starch-spermine and the oxidized starch are not toxic, even at 2 mg/mL. The starch-TEPA showed an average toxicity 14 ACS Paragon Plus Environment

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around 40 % and the starch-TREN was the most toxic. Regarding the MTT assays, the tendency was the same, but the sensitivity of this assay led to higher values: the starch-spermine can be considered as safe (cell viability around 80 %), the starch-TREN was toxic with a cell viability of 5 % at 1 mg/mL, the oxidized starch showed a cell viability of 55 % at 2 mg/mL and the starch-TEPA revealed an intermediary behavior, with a IC50 of 0.7 mg/mL. At 0.2 mg/mL, the starch-TEPA appeared safe with a cell viability > 80%. This difference in results between MTT and LDH assays on A549 cells may rely on the fact that the LDH activity assay is suitable for determining cytotoxicity by membrane damage but is not adapted for determining the extent of increased and decreased cell numbers due to, for example, cell cycle alterations without membrane damage.34 The cationic property of molecules is known to be cytotoxic toward cells.35 In our study, the starch-spermine had the lowest number of cationic amines, and was indeed the less toxic. However, the starch-TREN had an intermediate number of cationic amines and was the most cytotoxic. Indeed, the number of cationic charges has an influence but the molecular structure as well: the tertiary amines are more toxic than the secondary amine counterparts.35

Internalization of starch-TEPA/ATP in A549 cell line Synthesis of TEPA-starch-FITC

In order to follow the polyplexes by confocal microscopy, the fluorescein isothiocyanate was covalently coupled to starch according to R.B. Qaqish et al.36 The final compound was analyzed by FTIR and 1H NMR (Figure S4). As the starting molar ratio of starch:FITC was really low (50 : 1), it was difficult to finely see the presence of FITC, but by IR, excepting the characteristic peaks from starch molecule, additional absorption peaks between 1590 and 1450 cm-1 could be observed, which could refer to the C=C stretching from the benzene ring.37 In addition, a low intense signal was detected between 6 and 7 ppm in the 1H NMR spectra, which could fit with 15 ACS Paragon Plus Environment


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the ring protons of the FITC. The percentage of FITC grafted was quantified by measuring the intensity of the emitted fluorescence of each supernatant after three washings: 99 % of FITC was covalently coupled.

Formulation of TEPA-starch-FITC/ATP polyplexes

The formulation of TEPA-starch-FITC/ATP was done with the same protocol as described in Material and Methods: 1 mg/mL of modified fluorescent starch was mixed with ATP, at a N/P ratio of 2. The fluorescence of the suspension was easily observable, demonstrating again the covalent coupling of FITC (Figure S5). Polyplexes revealed a size of 356 ± 8.1 nm, a PdI of 0.08 and a zeta potential of -10.07 ± 0.54 mV.

Uptake studies

The uptake experiments of TEPA-starch-FITC/ATP polyplexes on A549 cells were investigated at 37°C and 4°C, with a concentration in TEPA-starch-FITC of 0.1 mg/mL and 0.2 mg/mL, and different times of internalization were studied: 1, 4 and 8 h (Figure 4 and S6). Polyplexes could be followed in green thanks to the FITC whereas cellular nucleuses were in blue (DAPI staining). At 37°C, when taken up by cells, polyplexes appeared as green spots in the median plane of the cells and accumulated along time within the intracellular compartments. After 1 h, polyplexes were located close to cell membrane. After 4 h, green spots seemed more dispersed inside the cytosol and after 8 h, this propagation was confirmed and the fluorescence was more pronounced. Some polyplexes could even be seen close to the nucleus. On the contrary, at 4°C, no polyplexes were internalized by the cells, even after 8 h. So TEPA-starch-FITC/ATP 16 ACS Paragon Plus Environment

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polyplexes penetrated through the cell wall of A549 cells via an energy-dependent pathway, most probably by endocytosis, and a simple diffusion seemed to be weak.38,39

1h

4h

8h

37°C

4°C

Figure 4. Confocal microscopy images after 1 h, 4 h and 8 h of incubation of A549 cells with TEPA-starch-FITC /ATP polyplexes. The suspension was incubated at a concentration of 0.1 mg/mL at 37 °C and 4°C. Blue: DAPI, nucleus. Green: FITC, polyplexes.

Biological activity

The biological activity of the different polyplexes studied in 3.2.5, starch-TEPA/ATP, starchTEPA/ddATP, starch-TEPA/ddCTP and starch-TEPA/ddGTP was determined on A549 cell line and compared with the free NAs at a concentration of 0.3 mg/mL. A MTT assay was done at a concentration of 0.2 mg/mL in starch-TEPA: considering the N/P ratio of 2 and a CE around 17 ACS Paragon Plus Environment


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100 %, the concentration in NAs was around 0.3 mg/mL (Figure 5). At this concentration, the modified starch could be considered as safe.

*

Figure 5. Box plot representing the biological activity of free ATP (0.3 mg/mL), ddATP (0.3 mg/mL), ddCTP (0.3 mg/mL) and ddGTP (0.3 mg/mL) and complexed with starch-TEPA (NP) at 0.2 mg/mL, containing a concentration of around 0.3 mg/mL of NAs (in considering 100 % of CE). (*) p-value < 0.01.

Regarding the free ATP, ddATP and ddCTP and their polyplexes, the biological activity was low (< 35 %), probably due to the cationic starch itself. The NAs, when incorporated into the DNA, causes premature chain termination and a limited passive intracellular diffusion is observed due to the hydrophilicity of the molecule and an ineffective intracellular conversion of these molecules into 5'-triphosphate.5 For the free ddGTP, around 20 % of cells were killed and for the polyplexes a biological activity of around 99 % was observed, that is statistically significant (p < 0.01). Regarding the size of the different polyplexes, large aggregates could be observed in the case of starch-TEPA/ddATP and starch-TEPA/ddCTP (PdI>0.9). These aggregates were probably not be able to be internalized by cells, explaining the low biological activity, unlike to starch-TEPA/ddGTP polyplexes (size of 633.0 ± 6.8 and a PdI

Design of Polyamine-Grafted Starches for ... - ACS Publications

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