Designing DendronâPolymer Conjugate Based Targeted Drug

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Designing Dendron-Polymer Conjugate Based Targeted Drug Delivery Platforms with a ‘Mix-and-Match’ Modularity Burcu Sumer Bolu, Bianka Golba, Nazli Boke, Amitav Sanyal, and Rana Sanyal Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00595 • Publication Date (Web): 14 Nov 2017 Downloaded from http://pubs.acs.org on November 15, 2017

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Bioconjugate Chemistry

1

Designing

Dendron-Polymer

Conjugate

Based

2

Targeted Drug Delivery Platforms with a ‘Mix-and-

3

Match’ Modularity

4

Burcu Sumer Bolu†, Bianka Golba†, Nazli Boke†, Amitav Sanyal†‡, Rana Sanyal*†‡

5

†

6

Istanbul, 34342, Turkey.

7

*Author to whom correspondence should be addressed; E-Mail: [email protected]

8

ABSTRACT

9

Polymeric micellar systems are emerging as a very important class of nano-pharmaceuticals due

10

to their ability to improve pharmacokinetics and bio-distribution of chemotherapy drugs, as well

11

as to reduce related systemic toxicities. While these nano sized delivery systems inherently

12

benefit from passive targeting through the enhanced permeation and retention effect leading to

13

increased accumulation in the tumor, additional active targeting can be achieved through surface

14

modification of micelles with targeting groups specific for over-expressed receptors of tumor

15

cells. In this project, non-toxic, biodegradable, and modularly tunable micellar delivery systems

16

were generated using two types of dendron-polymer conjugates. Either an AB type dendron-

17

polymer construct with 2K PEG or an ABA type dendron-polymer-dendron conjugate with 6K

Department of Chemistry and ‡Center for Life Sciences and Technologies, Bogazici University,

ACS Paragon Plus Environment

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1

PEG based middle block was used as primary construct; along with an AB type dendron-polymer

2

containing a cRGDfK targeting group to actively target cancer cells over-expressing αυβ3/αυβ5

3

integrins. A set of micelles encapsulating docetaxel, a widely employed chemotherapy drug,

4

were prepared with varying feed ratios of primary construct and targeting group containing

5

secondary construct. Critical micelle concentrations of all micellar systems were in the range of

6

10-6 M. DLS measurements indicated hydrodynamic size distributions varying between 170 to

7

200 nm. An increase in docetaxel release at acidic pH was observed for all micelles. Enhanced

8

cellular internalization of Nile red doped micelles by MDA-MB-231 human breast cancer cells

9

suggested that the most efficient uptake was observed with targeted micelles. In vitro

10

cytotoxicity experiments on MDA-MB-231 breast cancer and A549 lung carcinoma cell lines

11

showed improved toxicity for RGD containing micelles. For A549 cell line EC50 values of drug

12

loaded micellar sets were in the range of 10-9 M whereas EC50 value of free docetaxel was around

13

10-10 M. For MDA-MB-231 cell line EC50 value of free docetaxel was 6x10-8 M similar to EC50

14

of non-targeted AB type docetaxel doped micellar constructs whereas the EC50 value of its

15

targeted counterpart decreased to 5.5x10-9 M. Overall, in this comparative study, the targeting

16

group containing micellar construct fabricated with a 2kDa PEG based diblock dendron-polymer

17

conjugate emerges as an attractive drug delivery vehicle due to the ease of synthesis, high

18

stability of the micelles and efficient targeting.

19

INTRODUCTION

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Polymeric micelles are emerging as a promising class of drug delivery system to address diverse

21

challenges in the area of cancer therapeutics since they can be loaded with various drugs thereby

22

increasing their bioavailability, improving the pharmacokinetics and overall efficacy.1–5 In


2

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general, the low molecular weight of many of the chemotherapy agents results in their fast

2

clearance, thus decreasing the residence time in the body. Oftentimes, the hydrophobic character

3

of these drugs makes the addition of excipients like Cremophore®EL a necessity.6 While the

4

excipient increases the solubility of the hydrophobic drug through its emulsification, a variety of

5

toxic side effects have been noted which warrants search for safer alternatives. Combined with

6

the narrow therapeutic indices and added systemic toxicities of such formulations at effective

7

doses leading to increased mortality rates, conventional chemotherapy is far from perfect. To

8

overcome some of these challenges, utilization of nano-sized carriers of chemotherapy agents are

9

on the rise because of their potential to eliminate several of these disadvantages. For instance, to

10

improve efficacy and decrease prominent cardiotoxicity of anthracyclines, liposomal

11

formulations such as Doxil® are widely used in the clinic. In recent years, polymeric micelles

12

have been employed in the clinic as another class of nano-sized delivery agents.7 These are

13

expected to be efficient carriers due to their ability to deliver encapsulated drugs in high

14

concentrations at tumor sites, as well as to provide sustained release profiles, thereby

15

significantly improving the pharmacokinetics of the drug.8,9

16

Assembled from macromolecules with distinct hydrophobic and hydrophilic regions,

17

polymeric micelles are supramolecular assemblies possessing the ability to load various

18

hydrophobic drugs solo10,11 or in combination.12,13 Poly(ethylene glycol) (PEG) based polymers

19

are widely used as the hydrophilic micelle exterior rendering the nanoparticles water soluble and

20

enabling them to avoid or minimize interaction with plasma proteins due to their lack of surface

21

charge.14 Linear or hyperbranched hydrophobic polymeric domains encapsulating the drugs are

22

shielded by this protective layer15 from opsonization and unwanted interaction by

23

reticuloendothelial system (RES). The hydrophobic domains in these carriers also improve the


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1

stability of drugs by preventing their degradation under prolonged exposure to physiological

2

conditions.16,17 Using highly branched, symmetrical and well-defined polyester dendrons as the

3

hydrophobic component of micelles improves their critical micelle concentration (CMC) values,

4

and their biodegradability through hydrolytic or enzyme dependent degradation results in gradual

5

drug release.18,19

6

It is known that PEGylated micelles can circulate in plasma for prolonged periods of time

7

in stealth state escaping capture by mononuclear phagocyte system (MPS).20,21 Circulating nano-

8

therapeutic agents can accumulate in tumor tissue by Enhanced Permeation and Retention (EPR)

9

effect.22,23 Due to lack of a well-organized vasculature network and impaired lymphatic drainage,

10

tumor environment has rather heterogeneous and leaky morphology enabling accumulation of

11

drug loaded nanoparticles in tumor tissue in a size and shape dependent manner.24 Consequently,

12

macromolecules with hydrodynamic sizes large enough to escape renal filtration or RES can

13

diffuse through tumor vessel and accumulate there by EPR.20 This type of passive targeting

14

achieves retention of loaded cargo around tumor environment. However, to trigger cellular

15

uptake despite the anti-biofouling PEG based exterior, employment of ligands targeting over

16

expressed receptors on tumor cells and triggering endocytosis mechanisms is crucial. In

17

particular, Arg-Gly-Asp (RGD) sequence containing peptides are widely used since they are

18

known to target αυβ3/ αυβ5 integrin receptors that are over-expressed in various tumor cells and

19

tumor angiogenic vessels.25–27

20

In this study, a “mix and match” micellar drug delivery system was designed by

21

employing dendron-polymer conjugates (Scheme 1). As primary constructs, AB type diblock or

22

ABA type triblock dendron polymer conjugates were synthesized to constitute the bulk of the


4

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1

micelle. The first block (A) was composed of a hydrophobic biodegradable polyester dendron,

2

while the other block (B) was linear PEG polymer. Although the building blocks are quite

3

similar, subtle differences in the micellar aggregates formed using these building blocks can be

4

expected. For the AB-type dendron-polymer conjugates, it can be anticipated that dendritic parts

5

will contribute to the formation of micellar core leaving the linear PEG chains on the periphery.

6

On the other hand, for the ABA type construct, both of the dendron units would form the

7

micellar core while the middle hydrophilic PEG segment remains at the exterior. Such

8

differences in constraints could lead to variations in the display of the PEG chains on the surface

9

of these micelles. They can also manifest in changes in the size and stability of micellar

10

constructs obtained from AB or ABA type dendron-polymer conjugates. Moreover drug loading

11

capacities of these micelles and even their interaction with cells might be affected from these

12

differences. To render these micellar constructs suitable for active-targeting, dendron-polymer

13

conjugates bound to a cyclic peptide as a targeting ligand was used as a secondary construct. For

14

dendritic part of all constructs forming the hydrophobic core, an acetal protected generation-4

15

polyester dendron (G4OX) was utilized to obtain low CMC values. This dendritic segment acts

16

as an efficient hydrophobic drug reservoir. It is also acid labile which can lead to enhanced drug

17

release profiles through hydrolysis under acidic conditions. For the hydrophilic part linear PEG

18

polymers were used; 2 kDa for AB type primary construct, 6 kDa for ABA type primary

19

construct and 3.5 kDa for secondary construct bestowing excellent solubility along with size

20

commensuration of the hydrophilic block in all constructs. By mixing primary and secondary

21

constructs in different feed ratios, micelles with varying amounts of targeting groups were

22

obtained, and the effect of introducing the cyclic RGD peptide-conjugated construct on

23

properties from size and stability, to drug release and cellular internalization was investigated. A


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1

widely used anti-cancer drug docetaxel was encapsulated into the micelles and the change in

2

drug efficacy modulated by the micellar carrier was probed in vitro on two RGD-sequence

3

recognizing cancer cell lines, namely the MDA-MB-231, human breast cancer and A549, human

4

lung cancer. At the onset of the study, it is anticipated that an in depth understanding of the

5

subtle differences in the macromolecular building blocks composing these micellar constructs,

6

would allow for optimization towards an ideal construct. Moreover, the ‘mix and match’ nature

7

of the optimal construct would allow one to rapidly obtain a drug loaded, suitably targeted nano-

8

therapeutic system for various types of cancers.

9

10 11 12

Scheme 1. Illustration of ‘mix-and-match’ fabrication of drug loaded micellar constructs using

13

AB and ABA type dendron-polymer conjugates for active-targeting. Micelles can be localized

14

around tumor environment through passive targeting, followed by cell surface receptor mediated

15

active targeting.

16


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1 2 3

RESULTS AND DISCUSSION

4

Synthesis and Characterization of Dendron-Polymer Conjugates.

5

To construct micelles in a modular fashion with varying amounts of surface ligands, three

6

different dendron-polymer conjugates were synthesized. As primary constructs an AB type

7

dendron-PEG conjugate mPEG2K-G4OX (2K) and an ABA type dendron-PEG-dendron

8

conjugate G4OX-PEG6K-G4OX (6K) were synthesized to constitute the major component of

9

assembled micelles. The fourth generation hydrophobic dendron unit provides the driving force

10

for assembly of these conjugates when exposed to aqueous environments. As the secondary

11

construct, an AB type dendron-polymer conjugate, cRGDfK-PEG3.5K-G4OX (RGD), bearing

12

the integrin targeting cRGDfK group was synthesized. The targeting peptide was conjugated at

13

the end of the hydrophilic PEG chain so that it is displayed on the surface of the micelle. To

14

obtain the AB and ABA type primary constructs, a hydrophobic fourth generation polyester

15

dendron (G4OX) containing an alkyne group at its core was conjugated with azide functionalized

16

linear PEG polymers (2 kDa, 6 kDa) via the copper catalyzed azide-alkyne “Huisgen Click”

17

reaction (Scheme 2). The chemical composition of the dendron-polymer conjugates was verified

18

using 1H-NMR analysis (Figure 1), where apart from the expected proton resonances belonging

19

to the PEG and dendron components, the presence of the signal belonging to the proton on the

20

triazole ring (around 7.86 ppm) confirms conjugation though the triazole moiety. To obtain the

21

dendron-polymer conjugate containing the targeting group at the end of the hydrophilic PEG

22

chain, a hetero-bifunctional PEG (3.5 kDa) containing a NHS ester and an azide unit was utilized

23

(Scheme 3). First, the fourth generation polyester dendron was attached using the azide-alkyne


7


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1

click reaction, followed by the conjugation of the amine group containing cyclic RGD peptide to

2

the NHS-activated carboxylic acid group. After purification, the 1H-NMR spectrum of the

3

targeting ligand-containing dendron-polymer conjugate revealed the expected proton resonances

4

at 7.86 ppm arising from the triazole ring, and also displayed signals between 7.00-7.48 ppm

5

belonging to various protons on the cRGDfK fragment (Figure 2).

6

7 8

Scheme 2. Synthesis of primary constructs mPEG2K-G4OX and G4OX-PEG6K-G4OX.


8

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1 2

Figure 1. 1H-NMR of mPEG2K-G4OX and G4OX-PEG6K-G4OX. O O

O

O

O

O

O

O O O

O

O

O

O

O

O O

O

O O

O

O

O

O

O

O O

O O

N3

O

O

75

N

O O

o

THF, 40 C O

O

HN

O

O

O

HN

O

H 2N

O

O

HN

O

O

O

O

O O

O

DIPEA DMF, rt

O

NH O

O

O O O

O O

NH

HN O

O O

N N

N

O 75

OH

O N H

NH O

HN O

O O

O O

O

O O

O

O

H N

O

O

O

O

NH2

O

O

O

NHS-PEG3.5K-G4OX O

O

O

O

N

OH

O

O

O 75

NH O

HN

O

O

O

O

O

O O

O

O

O

O

cRGDfK-PEG3.5K-G4OX

O O

O

O

5

O

O

O

O

N

O

O

O

O

N N

O

O

O cRGDfK NH

O

O

O

O H N

O O

NH

O

O

O O

O

O

O

4

O O

O

O

O O NH 2

3

O

O

O

O O

O

O

O

Cu(I)Br, PMDETA

O O

O O

O

O O O O

O

O

O

O

O

O

O O

O

O

O

O

O

Scheme 3. Synthesis of targeting group bearing secondary construct cRGDfK-PEG3.5KG4OX.


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1 Figure 2. 1H-NMR of secondary construct cRGDfK-PEG3.5K-G4OX.

2 3 4

Generation and Characterization of Micelles Formed from Primary and Secondary

5

Constructs.

6

With the polymeric building blocks for fabricating the micelles at hand, two sets of micelles

7

(with and without the drug) were obtained using the primary constructs, 2K or 6K PEG-based,

8

along with the targeting ligand containing secondary construct at various weight percentages

9

(wt%) (Table 1). Micelles were formed via solvent evaporation method followed by dialysis.

10

Drug loading ranged between 9.86±0.25% and 10.21±0.21% and it was observed that the type of

11

primary construct or the weight percent of secondary construct did not affect final encapsulation

12

efficiency.


10

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1


Table 1. Micelle Compositions and Drug Contents.

2 3

Critical Micelle Concentrations. The CMC of these micelles were determined using a

4

fluorescent probe, namely, Nile red (NR). When in aqueous media, NR molecules form

5

excimers, quenching their signal, however once introduced into the hydrophobic core of

6

micelles, the dyes solubilize and an increase in peak intensity with a hypsochromic shift

7

observed in their emission spectrum. The CMC values of NR doped 2K and 6K micelle sets

8

prepared with varying weight ratios were investigated by tracking the blue-shifted NR peak

9

emission intensity at 636 nm (λex=550 nm) and determining the intersection of resulting

10

trendlines (Figure 3).


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1 2

Figure 3. Nile red emission spectra of 2K (A) and 6K (B) micelles. CMC values were identified

3

by intersection of two apparent linear fit trendlines ( denotes 100-0, denotes 90-10,

4

denotes 80-20 and denotes 60-40 micelles for both 2K and 6K micelle sets).

5

It was observed that the CMC values of 2K micelles were around 0.012 mg/mL, whereas the

6

CMC of ABA type 6K micelles were almost three times higher (0.033 mg/mL). But once these

7

values are converted to molar scale by converting milligram values of contributing primary

8

and/or secondary constructs while preparing individual micelles to moles, CMC of both micelles

9

types are quite similar (around 2.5x10-6 M) (Figure 4, Table 2). As seen in Figure 4B,

10

introduction of RGD bearing secondary construct had a slightly negative effect on both 2K and

11

6K micelles. Yet since CMC values were too close to each other and standard deviations were

12

notably high, this trend should be further supported by similar data from other micellar systems.


12

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1 2

Figure 4. CMC values of 2K and 6K micelles by mg/mL (A), molarity (B) representation.

3

Table 2. Tabulated CMC values of 2K and 6K micelles. Construct

CMC, mg/mL

CMC, [M]

2K 100-0

0.014 ±0.002

1.4E-06 ±8.3E-07

2K 90-10

0.011 ±0.001

2.4E-06 ±6.8E-07

2K 80-20

0.011 ±0.001

2.4E-06 ±5.4E-07

2K 60-40

0.012 ±0.001

2.3E-06 ±5.2E-07

6K 100-0

0.024 ±0.009

2.3E-06 ±1.7E-07

6K 90-10

0.039 ±0.007

3.6E-06 ±2.1E-07

6K 80-20

0.035 ±0.006

3.1E-06 ±2.6E-07

6K 60-40

0.033 ±0.006

2.6E-06 ±2.8E-07

4

Micelle Size and Stability towards Dilution. To study the hydrodynamic size of the micellar

5

constructs in aqueous environment, DLS measurements of micelles prepared at different feed

6

ratios and with or without drug encapsulation were undertaken (Figure 5).


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1 2

Figure 5. Hydrodynamic sizes from volume distribution of 2K and 6K micelle sets without or

3

with drug (D) encapsulation.

4

Table 3. Tabulated hydrodynamic size values from volume distributions of 2K and 6K micelle

5

sets without or with drug (D) encapsulation. Standard deviation (Std) and PDI (polydispersity

6

index) values were obtained from Zetasizer software by creating average results from 3 samples

7

for each case.

8 9

Interestingly, it was observed that on an average, the 6K micelles (118-139 nm) were slightly

10

smaller than 2K micelles (145-164 nm) without drug encapsulation (Table 3). After docetaxel

11

loading, the 2K micelles (168-198 nm) were smaller when compared to their 6K feed ratio


14

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1

counterparts (182 nm-226 nm). As expected, an increase in the micellar size was observed in all

2

cases upon loading the hydrophobic drug.

3

Introduction of the targeting peptide bearing secondary component to both 2K and 6K micelle

4

systems resulted in a slight increase in their size. A similar trend was observed for drug loaded

5

2K micelles. However, introduction of the secondary construct in increasing feed ratios resulted

6

in a decrease in micelle sizes for drug loaded 6K micelles. For drug loaded 2K and 6K micelles,

7

incorporation of secondary construct to the resulting assemblies led to a slight decrease in PDI

8

values which might indicate improved size distribution. However, the standard deviation values

9

suggests that the micellar aggregates are distributed over a wide size range (118-226 nm) and the

10

provided size values from volume distributions indicates the mean of each micelle population

11

(Figure SI 1-5).

12

Micelle integrity below CMC values was also investigated by diluting four types of micelles

13

to concentration of 0.005 µg/mL which coincides to over thousand fold dilution beyond CMC

14

values which were also indicated as dashed lines on Figure 6A. Notably, the integrity of micelles

15

was retained even under such harsh dilutions and PDI values remained near 0.2 (Figure 6B).

16

Only notable size change was observed for 2K 100-0 micelles at very high dilutions. The 6K

17

100-0 micelle set also displayed minor increase in size, yet neither of the targeting group

18

containing micelles (2K 60-40 nor 6K 60-40) exhibited any noticeable change in their size

19

(Figure 6A). Importantly, all micelles demonstrated prominent resistance against harsh dilutions

20

which makes these assemblies promising candidates for various applications.


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Page 16 of 43

1 2

Figure 6. Micelle (A) Z-average size (B) PDI values obtained after serial dilutions and (C)

3

corresponding dilution factors ( denotes 2K 100-0, denotes 2K 60-40, denotes 6K 100-0

4

and denotes 6K 60-40 micelles).

5


16

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1

Serum stability of the same set of micelles was investigated by FRET analysis using

2

previously established protocols.28 Effect of serum proteins triggering release from micelles was

3

monitored by the change of fluorescence signal intensities of FRET pair DiO and DiI at 501 and

4

565 nm. During serum incubation, release of encapsulated dyes would eliminate energy transfer

5

between donor DiO and acceptor DiI dye resulting in a decrease in FRET intensity at 565 nm

6

and an increase in DiO signal at 501 nm. Hence, micelle instability was recorded as a decrease in

7

the normalized FRET ratio I565/(I565 + I501) (Figure 7). Micelle solutions were incubated in FBS

8

or in 1x PBS for 30 h. Calculated FRET ratios suggested that both 2K and 6K micelles were

9

stable in 1x PBS for 30 h, whereas incubation in FBS resulted in a small decrease in FRET ratio

10

from 1 to 0.80-0.74 (Figure 7C). Also, increasing the amount of the targeting group containing

11

secondary construct in the micelles did not affect their stabilities in FBS notably. DLS profiles of

12

2K and 6K micelles were also determined at 30h (Figure SI 6). The significant increase in

13

hydrodynamic size and poor PDI values of 6K micelles after 30h FBS incubation indicated their

14

instability. However both 2K 100-0 and 2K 60-40 micelles stayed fairly stable in terms of their

15

hydrodynamic volume with a minor increase in their PDI values.


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Page 18 of 43

1 2

Figure 7. Normalized FRET ratios of 2K (A) and 6K (B) micelles in graphical representation.

3

Change in FRET ratios was monitored in 1XPBS and FBS throughout 30 h at 37°C. Final FRET

4

ratios at 30 h are given in table (C). ( denotes 100-0 in PBS, denotes 60-40 in PBS,

5

denotes 100-0 in FBS:PBS (1:1) and denotes 60-40 micelles in FBS:PBS (1:1) for both 2K

6

and 6K micelle sets.)


18

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1

The two types of drug loaded 2K and 6K micelles were also investigated using TEM to

2

determine their size in dry form. Even though the sizes of drug loaded 2K and 6K micelles

3

determined via DLS varied by 60 nm (Figure 5B), once dried their size difference decreased

4

dramatically. In dry form it is highly possible that 2K and 6K micelles shrink considerably.

5

Consequently, measured mean diameters of micelles with TEM were considerably smaller than

6

their solvated states (Figure 8). In TEM analysis, the micelles could also appear smaller because

7

of the lack of visibility of the PEG chains due to their poor contrast. The smaller core size might

8

render them suitable for treatment against denser tumor types as well. However, to determine

9

their optimum target tumor tissue they must be tested in vivo first.

10

11 12

Figure 8. Dry micelles and measured mean micelle diameter values from TEM (Scale bar 200

13

nm).

14

pH Dependent Docetaxel Release from Micelles. Release profile of encapsulated drug is

15

especially important for micellar systems since they are generally prone to early release due to

16

lack of more durable covalent bonds stabilizing the assembly. To determine drug release profiles,

17

docetaxel release at pH 7.4 and pH 4.8 was monitored using LC-MS. Since the core of 2K and

18

6K micelles were composed of biodegradable polyester G4 dendrons, release in acidic


19


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Page 20 of 43

1

environment was expected to be higher than those at physiological buffer system. Furthermore,

2

to check the effect of secondary construct on drug release, a set of 2K and 6K micelle (100-0 and

3

60-40) with and without the targeting ligand component were compared. After 30 hours of

4

incubation at 37 °C, it was observed that 2K micelles were more stable in terms of drug release

5

at 7.4. In the absence of secondary construct, docetaxel release percent reached to only

6

18.6±1.7% for 2K 100-0 D micelles, whereas it was almost double for the 6K 100-0 D micelles

7

with 34.1±0.5% (Figure 9). Introduction of the RGD bearing secondary construct increased

8

docetaxel release for both 2K and 6K at pH 7.4. On the other hand, drug release was notably

9

increased for all micelle types at pH 4.8 as expected and reached to a maxima of 68% for both

10

2K and 6K 60-40 D micelle sets in 30 hours. The increase in percent drug release indicates that

11

employing a secondary construct, even with the same core structure, can alter release profiles of

12

micelle systems formed from AB and ABA type polymers. However, the increased release at

13

acidic pH combined with the targeting effect of RGD group at their periphery suggests the

14

suitability of these micellar constructs for delivery applications. To demonstrate the acid labile

15

character of the polyester dendrons and hence the micelles, DLS profiles of 2K micelles

16

incubated in PBS buffer (pH 7.4) or in acetate buffer (pH 4.8) at 37 °C were determined (Figure

17

SI 7). Hydrodynamic sizes of micelles incubated at physiological pH were stable after 24h of

18

incubation. On the other hand incubation in acidic buffer resulted in a notable increase in z-

19

average values and slight increase in PDI values increased even after 10h. After 24h micelle

20

integrity was severely disrupted reflected by poor z-average and PDI values. These profiles

21

indicate a time dependent gradual disruption of micelles at acidic environments.


20

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1 2

Figure 9. Percent docetaxel release profiles from 2K (A) and 6K (B) micelles in graphical

3

representation. Final release percentages at 30 h are given in Table (C) ( denotes 100-0 D

4

micelles incubated at pH 7.4, denotes 60-40 D micelles incubated at pH 7.4, denotes 100-0

5

D micelles incubated at pH 4.8 and denotes 60-40 D micelles incubated at pH 4.8 for both 2K

6

and 6K micelle sets).


21


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Page 22 of 43

1

In vitro Cellular Internalization of Targeted and Non-targeted Micellar Constructs. To

2

assess cellular internalization, the 2K and 6K micelles with and without targeting group were

3

incubated with MDA-MB-231 human breast cancer cells. In order to visualize internalization of

4

the cargo, micelles were doped with a hydrophobic dye, namely, Nile red. In comparison to the

5

control well, where NR was added to cell containing media in the absence of any micelles, there

6

was a visible increase in the NR intensity in the cells treated with all micelle types after 1 h of

7

incubation (Figure 10). There was a minute difference between 100-0 and 60-40 micelles, the

8

latter showing more internalization in comparison. After 3 h, NR signal considerably increased

9

in all wells except the control, indicating cell internalization of NR was improved dramatically

10

once encapsulated into micelles and the uptake increased over time. Here, it is important to note

11

that NR signal at both 60-40 micelles rose dramatically highlighting the significance of the RGD

12

targeting group for internalization through active targeting. A change in cell shape histology was

13

also notable at 3 h for 60-40 micelles; possibly due to interaction of RGD on micelles with

14

integrin receptors on the cell surfaces, the shape of MDA-MB-231 cells became rounder instead

15

of their more spread counterparts. Also a slightly increased NR intensity was observed for 2K

16

micelles in contrast to 6K micelles, thus hinting higher internalization efficiency with the AB

17

type dendron-polymer conjugates.


22

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1 2

Figure 10. Nile red doped 2K and 6K micelles and Nile red dye only as control were incubated

3

for 1 h and 3 h with MDA-MB-231 cells. Merged images were generated by overlapping DAPI

4

channel (blue) with NR channel (red) (Scale bar 50µm).

5


23


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Page 24 of 43

1

The cellular internalization of the NR doped targeted and non-targeted micelles were also

2

investigated using flow cytometry. To further emphasize the RGD dependent internalization,

3

cRGDfV was employed acting as a competitive inhibitor of conjugated cRGDfK for integrin

4

binding. It is known that cRGDfV has higher binding affinity towards the integrin domains on

5

cell surfaces.29,30 After 1 h, a significant increase in NR internalization was observed for all

6

micelles, however 2K micelles showed higher mean fluorescence intensity (MFI) values

7

compared to 6K counterparts (Figure 11). Besides NR internalization was higher for both the 60-

8

40 micelles compared to 100-0 micelles, where the later ones were devoid of RGD moieties on

9

micelle surface. Furthermore, addition of cRGDfV to cell media in combination with 60-40 NR

10

micelles resulted in a decrease in NR intensity, with values comparable to the 100-0 NR

11

counterparts, indicating that the increased internalization was due to interaction of RGD on

12

secondary construct with the integrins on MDA-MB-231 cell surfaces. After 3 h, the NR MFI

13

values increased further (Figure 11C) thus highlighting the time dependent gradual manner of

14

internalization.


24

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1 2

Figure 11. Nile red cell internalization histograms by flow cytometry of doped micelles (A)

3

2K, (B) 6K for 1 h, and (C) MFI values for cells incubated for 1 and 3 h with micelles or NR

4

only. To display RGD dependency, cRGDfV inhibitor was added as a competitor to indicated

5

samples.

6 7

In vitro Cellular Viability Assays. Cellular toxicity levels of 2K and 6K micelles were

8

investigated on two cell lines expressing one or more of RGD recognizing integrins, namely

9

MDA-MB-231 breast and A549 lung cancer cell lines.34-36 After 48 h continuous incubation of

10

blank micelles with cells, cell viabilities were determined via CCK-8 assay (Figure 12). MDA-

11

MB-231 breast cancer cell line showed minimal growth inhibition even at 1 mg/mL polymer

12

concentration. Micelle cytotoxicity was also well compensated by A549 lung carcinoma cell


25


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Page 26 of 43

1

line; varying between 62 to 100% at 1 mg/mL polymer concentration, cell viability was

2

unhindered at concentrations of 0.5 mg/mL and lower. There were no trending differences

3

between 2K and 6K micelles or at different feed ratios of secondary construct, valid for both cell

4

lines. For instance, 2K micelles showed improved viability in comparison to 6K set for MDA-

5

MB-231. On the other hand viability profiles of 6K were slightly better for A549 cells at certain

6

concentrations. Similarly, addition of RGD containing secondary construct did not result in a

7

distinct cytotoxicity increase at the tested cell lines. Despite the 48 h long incubation time with

8

blank micelle solutions, all tested cell lines exhibited significant cell viability, demonstrating

9

their potentials as drug delivery platforms.

10 11

Figure 12. Cell Viabilities of empty 2K and 6K micelles on A549 (A), and MDA-MB-231 (B)

12

cell lines after 48 h of continuous incubation.


26

Page 27 of 43

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1

In vitro Cell Viability Assay with Docetaxel loaded Micelles. Four types of docetaxel

2

encapsulated micelles were incubated with A549 and MDA-MB-231 cell lines for 48 h

3

continuously, resulting cell viabilities were compared to controls and percent viabilities were

4

calculated (Figure 13). For all cell lines, cellular toxicities of 2K and 6K 100-0 D micelles were

5

similar to each other at almost all concentrations. Introduction of RGD to 2K and 6K systems

6

improved cytotoxicity for both cell lines and at practically all concentrations. The decrease in

7

cell viabilities was more prominent with MDA-MB-231 cells especially after 10-8 M drug

8

concentrations (Figure 13B). For MDA-MB-231 at 10-6M, cell viability of 2K 60-40 D micelles

9

decreased to 64% whereas 2K 100-0 D micelles resulted in 70% viability similarly only 67% of

10

6K 60-40 D treated cells were alive where this ratio was around 75% for 6K 100-0 D micelles.

11

For A549, 2K 60-40 D showed a notable decrease in cell viability at most concentrations (max

12

14.4% at 10-7 M ) compared to non-targeted 2K 100-0 D micelles but change with 6K 60-40 D

13

micelles were not too distinct. Still, docetaxel showed best overall cytotoxicity on A549 cell line

14

and construct itself induced minimal cytotoxicity to A549 cells (Figure 13A). Compared to free

15

drug values, EC50 values showed an increase for all micelle types with A549 cell lines (Table 4).

16

This type of increase can be expected owing to the gradual release dynamics of drug molecules

17

from micelles. On the other hand, MDA-MB-231 cell line showed almost no change in EC50

18

values compared to docetaxel for 2K 100-0 D micelles whereas a minor decrease was obtained

19

with the 2K 60-40 D set, indicating the positive effect of RGD targeting on cellular toxicity.

20

EC50 values of 6K set was slightly higher in comparison with improved EC50 value from RGD

21

containing 6K 60-40 D micelles which was in the range of free docetaxel in this case.


27


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Page 28 of 43

1 2

Figure 13. Cell viabilities of docetaxel loaded 2K and 6K micelles on A549 (A), and MDA-MB-

3

231 (B) cell lines after 48 h of continuous incubation.


28

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1

Table 4. EC50 values of micelle encapsulated or free docetaxel for A549 and MDA-MB-231

2

lines after 48 h of continuous incubation.

3 4

To emphasize the impact of RGD on cellular internalization, a pulse chase experiment was

5

performed with MDA-MB-231 cell line; in this case incubation time of drug and drug doped

6

micelles were shortened to 2 h followed by 22 h incubation in fresh media. If secondary

7

construct containing micelles had any positive effect on cell binding and hence internalization of

8

encapsulated drugs, a change in cell viabilities of 60-40 micelle set was expected. Indeed, a

9

decrease in cell viability with respect to free docetaxel treated cells was observed with 60-40

10

micelles with pulse chase treatment (Figure 14B). For 2+22h treatments cell viability at 10-5 M,

11

was 69.9 % for 2K 60-40 and 74.2 % for 6K 60-40 micelles where the cell viabilities for 100-0

12

counterparts were 85.1 % for 2K 60-40 and 87.7 % for 6K 60-40 micelles. Free drug on the other

13

hand showed almost no growth inhibition and 100-0 D micelles achieved only slight

14

cytotoxicities even at the highest concentration.


29


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Page 30 of 43

1 2

Figure 14. Cell viabilities of docetaxel loaded 2K and 6K micelles on MDA-MB-231 cell line

3

after (A) 48 h of continuous incubation, and (B) 2+22 hours of pulse chase incubation.

4 5

CONCLUSIONS

6

Two types of micellar systems were generated using polymer-dendron conjugates: an AB type

7

diblock construct using a 2K PEG and an ABA type triblock construct using a 6K PEG as the B

8

block. Both primary constructs contained a fourth generation polyester dendron as the

9

hydrophobic component (block A). The micellar system was designed such that varying amount


30

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1

of targeting ligands could be introduced on their surface. This was accomplished by the

2

introduction of a secondary dendron-polymer construct bearing a cyclic RGD targeting group.

3

Fabricated micelles possessed very low CMC values (ca. 10-6 M), and they preserved their

4

stability on further dilutions. Likewise, serum stability experiments indicated that they were

5

stable in FBS and able to escape burst release which is a pronounced problem for most micelle

6

systems. Importantly, the drug loaded micelles exhibited pH related docetaxel release

7

emphasizing the enhanced delivery of their cargo near acidic tumor environment or once

8

internalized into targeted cells. Their hydrodynamic sizes were around 200 nm which is suitable

9

for benefiting from EPR effect except maybe for dense tumor types. However, TEM images

10

indicated that their dry size was much smaller, around 25 nm. It was observed that the

11

cytotoxicity of docetaxel loaded micelles improved with higher RGD containing 60-40 micelle

12

sets. Pulse-chase experiments highlighted the importance of micellar drug delivery; particularly

13

60-40 micelles showed improved cytotoxicity where free drug itself failed to act at these

14

conditions.

15

Overall, the 2K micelles showed slightly better results and can be chosen as a preferred

16

candidate for subsequent applications, due to their facile synthesis, when compared with the

17

ABA type conjugates. It is also important to note that this modular micellar drug delivery

18

construct can be used with different types of cancer by simply changing the targeting group on

19

secondary construct and the encapsulated drug. This ‘mix and match’ palate for optimizing

20

effective drug delivery to various types of cancers would make these polymeric delivery systems

21

attractive candidates in the field.

22


31


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Page 32 of 43

1 2

EXPERIMENTAL SECTION

3

Materials. Poly(ethylene) glycol (PEG) derivatives were purchased from Fluka. Dowex-H, 2,2-

4

bis(hydroxymethyl) propionic acid (bis-MPA), propargyl alcohol were purchased from Alfa

5

Aesar. Azide PEG succinimidyl carboxymethyl ester (N3-PEG3.5kDa-NHS) was purchased

6

from JenKem Technology (USA). All amino acids required for cRGDfK synthesis was

7

purchased from Protein Technologies Inc. (USA). All solvents were purchased from Merck, all

8

dyes including Nile red (NR), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate

9

(DiI) and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) were obtained from Aldrich. Bis-

10

azido functionalized 6kDa PEG, azido functionalized 2kDa monomethoxy PEG and generation

11

4 bis-MPA polyester dendron (G4OX) and cRGDfK were synthesized and characterized

12

according to formerly reported procedures.31–33

13

Instrumentation. The monomer and copolymers were characterized by 1H-NMR spectroscopy

14

(Avance III HD, 400MHz, Bruker, USA) and attenuated total reflection Fourier transform

15

infrared (ATR-FT-IR) spectroscopy (Nicolet 380, Thermo Scientific, USA). Synthesis of

16

cRGDfK was achieved through solid phase peptide synthesis (SSPS, PS3 Peptide Synthesizer,

17

Protein Technologies Inc., USA). Fluorescence intensity of Nile red (NR) is measured with a

18

fluorescence spectrometer (Varian Cary Eclipse, Agilent, USA). Wet size of the micelles was

19

monitored with dynamic light scattering (DLS, Zetasizer ZS, Malvern, USA). Measurements

20

were done at 37 °C after 2 min equilibration time with 173 ° backscatter detection angle. Their

21

dry form was analyzed on holey carbon film coated 300 mesh Cu grid (Pacific Grid Tech, USA)

22

using transition electron microscopy (TEM) with an accelerating voltage of 5kV (LVEM5


32

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1

Electron Microscope, Delong Instruments, USA). Docetaxel was quantified by liquid

2

chromatography–mass spectrometry (Shimadzu LCMS-2020, Japan) analysis using a C-18

3

column (5 µm 150 × 4.6 mm). Mobile phase consisted of dH2O and HPLC grade acetonitrile

4

(ACN) containing 0.05% trifluoroacetic acid with a flow rate of 0.5 mL/min and using the

5

following gradient; LC: 0-3 min, 50% ACN; 3.01 min, 50% ACN; 8 min, 95% ACN; 8.01-11

6

min 50% ACN MS single ion mode. Docetaxel peak was detected through its mass signal at

7

807.9 m/z (single ion mode) along with its PDA signal at 254nm with elution time of 6.8 min.

8

Docetaxel standards as well as docetaxel containing samples were prepared in 1:1 dH2O: ACN

9

with 0.05% trifluoroacetic acid final concentration. MilliQ Water Purification System (Merck

10

Millipore, USA) provided ultra-pure water. The absorbance values (450 nm) were recorded with

11

a plate reader. (Multiscan FC, Thermo Scientific, USA). Time dependent dye internalization

12

experiments were done with an inverted fluorescence microscope (Zeiss Observer A1 equipped

13

with AxioCam MRc5). Both dye internalization and competitive binding was studied by flow

14

cytometry through Red-B filter (965/50nm) with a flow rate of 500 particles/µL (Guava

15

EasyCyte, Merck Millipore, USA), and results were processed on guavaSoft software.

16

Synthesis and Characterization of G4OX. Fourth generation polyester dendron with an alkyne

17

group at the focal point was synthesized as previously reported in literature.25

18

Synthesis of Primary Constructs mPEG2K-G4OX and G4OX-PEG6K-G4OX. Synthesis of

19

ABA and AB type dendritic linear diblock and triblock copolymers bearing G4OX dendron was

20

adapted from literature with minor modifications.32–34 Alkyne core containing G4 dendron (200

21

mg, 0.0946 mmol) was reacted with mPEG2K-N3 (126.08 mg, 0.0630 mmol) or N3-PEG6K-N3

22

(189.13 mg, 0.0315 mmol) in the presence of PMDETA (13.31 µL, 0.0630 mmol) and Cu(I)Br

23

(9.04 mg, 0.0630 mmol) in THF (3.0 mL) to yield mPEG2K-G4OX (2) (176.4 mg, 68% yield)


33


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Page 34 of 43

1

and G4OX-PEG6K-G4OX (3) (209.6 mg, 65% yield), respectively (Scheme 2). 1H NMR of

2

mPEG2K-G4OX (400MHz, CD3OD, δ, ppm): 7.86 (s, 1H), 5.23 (s, 2H), 4.54 (t, J=10 Hz, 2H),

3

4.28 - 4.19 (m, 60 H), 3.80 - 3.40 (s, 172 H), 3.37 (s, 3H), 1.41 (s, 24H); 1.35 (s, 24H); 1.30 (s,

4

6H), 1.28 (s, 12H), 1.14 (s, 24H) (Figure 1). 1H NMR of G4OX-PEG6K-G4OX (400MHz,

5

CD3OD, δ, ppm): 7.86 (s, 2H), 5.23 (s, 4H), 4.54 (t, J=10 Hz, 4H), 4.28 - 4.19 (m, 120 H), 3.80

6

- 3.40 (s, 756 H), 1.41 (s, 48H); 1.35 (s, 48H); 1.30 (s, 12H), 1.28 (s, 24H), 1.14 (s, 48H) (Figure

7

1).

8

Synthesis of Secondary Construct

9

Synthesis of cRGDfK by SSPS. cRGDfK synthesis was achieved using solid phase peptide

10

synthesis methodology based on literature.31 The peptide was characterized in LC-MS through

11

its retention time and expected and observed [M+H] value of 604.

12

Synthesis of NHS-PEG3.5K-G4OX. Synthesis of AB type dendritic linear diblock copolymers

13

bearing G4OX dendron was adapted from literature with modifications.32–34 Alkyne core

14

containing G4 dendron (200 mg, 0.0946 mmol) was reacted with N3-PEG3.5K-NHS (220.50 mg,

15

0.0630 mmol) in presence of N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA) (13.15

16

µL, 0.0630 mmol) and Cu(I)Br (9.04 mg, 0.0630 mmol) in THF (3.0 mL) resulting in 240.51

17

mg product with 68% yield (Scheme 3). 1H NMR of NHS-PEG3.5K-G4OX (400MHz, CD3OD,

18

δ, ppm): 7.86 (s, 1H), 5.23 (s, 2H), 4.54 (t, J=10 Hz, 2H), 4.28-4.19 (m, 60 H), 3.80-3.40 (s, 300

19

H), 2.84 (s, 4H), 1.41 (s, 24H); 1.35 (s, 24H); 1.30 (s, 6H), 1.28 (s, 12H), 1.14 (s, 24H).

20

Synthesis of cRGDfK-PEG3.5K-G4OX. N-Hydroxysuccinimide activated NHS-PEG3.5K-

21

G4OX (200 mg, 0.0363 mmol) was conjugated to cRGDfK (26.27 mg, 0.0436 mmol) from its

22

lysine residue in presence of N,N-diisopropylethylamine (DIPEA) (63.23 µL, 0.363 mmol) in

23

dimethylformamide (DMF, 2.5 mL) at room temperature in 48h. Resulting conjugate was


34

Page 35 of 43

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1

purified by dialysis in water for 1 day. cRGDfK-PEG3.5K-G4OX was obtained as a yellowish

2

powder via lyophilization (Scheme 3). 1H NMR of cRGDfK-PEG3.5K-G4OX (400MHz,

3

DMSO, δ, ppm): 7.73 (s, 1H), 7.48 - 7.00 (m, 5H), 5.17 (s, 2H), 4.50 (t, J=5 Hz, 2H), 4.30 - 3.6

4

(m, 60 H), 3.60 - 3.40 (s, 300 H) , 1.34 (s, 24H); 1.22-1.13 (m, 45H); 1.01 (s, 24H) (Figure 2).

5

Micelle Preparation. Four types of mix and match micelles were prepared using different

6

weight ratios of primary constructs mPEG2K-G4OX (2K) or G4OX-PEG6K-G4OX (6K) and

7

cRGDfK-PEG3.5K-G4OX. For preparation of micelles with and without drug, solvent

8

evaporation method was employed; briefly copolymer stock solutions (10 mg/mL) in methanol

9

were made. By combining required volumes from primary and secondary polymer stock

10

solutions exact amounts of dissolved polymers with certain weight ratios were distributed into

11

glass vials. After addition of water, methanol in vials was evaporated via rotary evaporation at 30

12

°C.

13

Loading of Docetaxel into Micelles. For drug loading, docetaxel stock solutions and polymer

14

solutions were prepared in methanol (10 mg/mL) and exact volumes of drug stock solution were

15

added onto polymer solution to obtain different polymer to drug weight ratios. After combination

16

of polymer and drug solutions in methanol they were vortexed briefly. Water was added into

17

polymer drug solution and to obtain micelles methanol was evaporated at 35 °C via rotary

18

evaporation under reduced pressure. To remove excess drug molecules, prepared micelles were

19

dialyzed in water for 2 days in sink conditions. Removal of free drug and drug precipitates were

20

tracked by LC-MS and DLS respectively.

21

Determination of Docetaxel Content. 100 µL of docetaxel loaded micelles (with polymer

22

concentration of 1 mg/mL) were lyophilized to determine their dry weight. Additionally, 100 µL


35


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Page 36 of 43

1

of the same micelle solutions were dissolved in 0.9 mL ACN for LC-MS analysis to quantify

2

their docetaxel content employing docetaxel calibration curves.

3

Determination of Critical Micelle Concentrations. Four set of NR doped micelles (2K 100-0,

4

2K 60-40, 6K 100-0 and 6K 60-40) were prepared by solvent evaporation method. Polymer stock

5

solutions containing primary and secondary constructs with desired weight ratios were prepared

6

in methanol (10 mg/mL). Using these stock solutions, serial dilutions were made in methanol and

7

solutions containing polymers with final concentrations ranging between 1 mg/mL to 3x10-5

8

mg/mL were obtained individually. Nile red solution in methanol (50 µL, 0.1mg/mL) was added

9

into each vial and after addition of deionized water (3 mL), methanol was evaporated.

10

Fluorescence measurements were taken with excitation wavelength of 550 nm and emission

11

spectra between 575 and 700 nm were recorded for NR doped micelle samples. By measuring

12

the blue shifted peak at 636 nm (660 nm in aqueous environment) CMC values were

13

calculated.35,36

14

Determination of Micelle Hydrodynamic Size and Dilution Stability by DLS. Eight set of

15

micelles at different concentrations (higher than CMC) with or without drug were prepared by

16

solvent evaporation method; 2K 100-0, 2K 90-10, 2K 80-20, 2K 60-40, 6K 100-0, 6K 90-10, 6K

17

80-20, and 6K 60-40. Their effective size diameters were determined at 37 °C. To monitor the

18

effect of dilution on micelle stability, prepared micelles were diluted up to 0.005 µg/mL (lower

19

than CMC) and DLS measurements were repeated.

20

Determination of Micelle Size by TEM. Four set of drug loaded micelles were prepared by

21

solvent evaporation method; 2K 100-0 D, 2K 60-40 D, 6K 100-0 D and 6K 60-40 D. Micelle

22

solutions (0.01 mg/mL) were placed onto 300 mesh copper grids (Pacific Grid Tech, USA) and


36

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1

air dried before image acquisition without any further staining. Mean size of micelles were found

2

by measuring at least 100 micelles with the Q-Capture Pro7 software.

3

pH Dependent Docetaxel Release from Micelles. Four set of drug loaded micelles (10 mg/mL)

4

were prepared by solvent evaporation method; 2K 100-0, 2K 60-40, 6K 100-0 and 6K 60-40.

5

Stock micelle solutions were diluted with acetate buffer (pH 4.8) and phosphate buffer (pH 7.4)

6

separately with final concentrations being 5 mg/mL. Final micelle solutions were placed into

7

dialysis bags (mwco 1000) which were immersed in 1 L of buffer solutions and incubated at 37

8

°C for 10 days. Aliquots (1 mL) were collected at different time points and released docetaxel

9

was quantified by LC-MS analysis.

10

Investigation of Serum Stability of Micelles using FRET. Four set of micelles were prepared

11

by solvent evaporation method; 2K 100-0 F, 2K 60-40 F, 6K 100-0 F and 6K 60-40 F containing

12

1 % w/w Förster resonance energy transfer (FRET) dye pair DiI and DiO. As previously reported

13

in literature,28 serum stability profiles of micelles were determined by the decrease in FRET

14

signal. Micelle solutions diluted 1:1 with FBS were incubated at 37 °C for 2 days and excited at

15

484 nm, and their emission intensities were recorded at 501 nm (I501) and 565 nm (I565) for DiO

16

and DiI respectively at predetermined time points. The change in FRET ratio, I565/(I565 + I501),

17

was calculated and normalized to time zero.

18

Cell Lines. A549 and MDA-MB-231 cell lines were purchased from ATCC (LGC Standards,

19

Germany) and grown according to manufacturer’s culture methods requirements. Cells were

20

cultivated at 37 °C in a humidified atmosphere of 5 % CO2.

21

Cytotoxicity Assay. Cell Counting Kit-8 (CCK-8, Fluka) was used to determine cell viability.

22

A549 or MDA-MB-231 cells were seeded into 96-well plates (5000 cells/well) in quadruplicates.


37


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Page 38 of 43

1

After incubation for 12 h, cells treated with conjugates, free drug or polymer only controls and

2

kept with the corresponding solutions for 48 h. For free drug treatments, docetaxel stock solution

3

was prepared in DMSO (10-3 M) so that free docetaxel dissolved completely. Serial dilutions

4

were made using cell media where final DMSO concentration was kept below 0.5 % (V/V) for

5

all dilutions. For pulse chase experiment, cells were incubated with drug containing solutions

6

which was replaced with fresh media after 2 h. After 22 h, CCK-8 solution was added to into

7

each well to culture for 4 h. The absorbance was recorded at 450 nm with a plate reader. Cell

8

media only treated cells were used as controls to calculate percent viability. EC50 values were

9

calculated from percent viability versus concentration response graphs by nonlinear regression

10

analysis.

11

Cellular Internalization of NR doped Micelles. Four set of NR doped micelles were prepared

12

by solvent evaporation method; 2K 100-0 NR, 2K 60-40 NR, 6K 100-0 NR and 6K 60-40 NR.

13

MDA-MB-231 cells seeded onto 24 well plates were treated with 1 % (w/w) NR containing

14

micelle solutions with 0.1 mg/mL final concentration. NR content of the loaded micelles was

15

determined via fluorescence spectroscopy to ensure similar dye loading levels. As control

16

samples, same amount of NR was added into wells. Cells were then incubated for 1, 3 and 24 h

17

at 37 °C, followed by fixing with 4 % formaldehyde after DAPI staining and fluorescence

18

images were taken via inverted fluorescence microscope.

19

cRGDfK Dependent Cell Binding Assay. Four set of NR doped micelles were prepared by

20

solvent evaporation method; 2K 100-0, 2K 60-40, 6K 100-0 and 6K 60-40. MDA-MB-231 cells

21

seeded onto 24 well plates were treated with 2.5 % (w/w) NR containing micelle solutions with

22

0.1 mg/mL final concentration. As control samples, same amount of NR was added into wells.

23

Also free cRGDfV, a cyclic integrin-binding peptide possessing higher binding affinity to RGD


38

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1

specific integrins compared to RGD was added as an inhibitor into another set of wells. Cells

2

were then incubated for 1 and 3 h at 37 °C. After each treatment, cells were trypsinized and NR

3

intensities were determined by flow cytometry.

4 5 6

ASSOCIATED CONTENT

7

AUTHOR INFORMATION

8

Corresponding Author

9

*E-mail: [email protected]. Phone: (90) 212 359 4793.

10

Author Contributions

11

Manuscript was prepared through contributions from all authors.

12

NOTES

13

The authors declare no competing financial interest.

14 15

ACKNOWLEDGMENT

16 17

We would like to thank Bogazici University Research Fund (BAP Project No: 14B05P6) for

18

funding. Also we thank the Ministry of Development of Turkey (Project 2009K120520) for their

19

contribution to the infrastructure. B.S.B. and B.G. thank The Scientific and Technological

20

Research Council of Turkey (TUBĐTAK) for graduate student fellowship from the TUBĐTAK-

21

BĐDEB 2211 and BĐDEB 2210 programs.


39


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Page 40 of 43

1 2

SUPPORTING INFORMATION

3

Supporting Information is available free of charge via the Internet at http://pubs.acs.org

4

containing histograms and tables with exact size for empty and drug loaded 2K and 6K micelles

5

from DLS; histograms and tables for 2K and 6K micelle stability tests in 10% FBS and acidic

6

pHs from DLS; result of flow cytometry experiment for displaying effect of secondary construct

7

ratio on MDA-MB-231 cell internalization.

8

ABBREVIATIONS

9

Acetonitrile (ACN), cell counting kit 8 (CCK-8), critical micelle concentration (CMC), 1,1′-

10

dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine

11

dioctadecyloxacarbocyanine perchlorate (DiO), N,N-diisopropylethylamine (DIPEA), dynamic

12

light scattering (DLS), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 4’,6-diamidino-

13

2-phenylindole (DAPI), effective concentration (EC50), enhanced permeation and retention

14

(EPR), fetal bovine serum (FBS), Förster resonance energy transfer (FRET), liquid

15

chromatography mass spectrometry (LC-MS), N-hydroxysuccinimide (NHS), Nile red (NR),

16

mean fluorescence intensity (MFI), phosphate buffered saline (PBS), PDI (polydispersity index),

17

poly(ethylene

18

reticuloendothelial system (RES), Arg-Gly-Asp (RGD), tetrahydrofuran (THF).

glycol)

(PEG),

perchlorate

(DiI),

N,N,N′,N′′,N′′-pentamethyldiethylenetriamine

3,3′-

(PMDETA),

19 20

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