Size-Transformable, Multifunctional Nanoparticles from

Jan 15, 2019 - †Department of Coatings and Polymeric Materials and ‡Materials and Nanotechnology Program, Department of Physics, ⊥Department of ...
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Controlled Release and Delivery Systems

Size-transformable, Multi-functional Nanoparticles from Hyperbranched Polymers for Environment-specific Therapeutic Delivery Priyanka Ray, Lina Alhalhooly, Arnab Ghosh, Yongki Choi, Sushanta K Banerjee, Sanku Mallik, Snigdha Banerjee, and Mohiuddin Quadir ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01608 • Publication Date (Web): 15 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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ACS Biomaterials Science & Engineering

Size-transformable, Multi-functional Nanoparticles from Hyperbranched Polymers for Environment-specific Therapeutic Delivery Priyanka Ray

a,

Lina Alhalhooly b, Arnab Ghosh

Sushanta Banerjee Mohiuddin Quadir a

c,d,

Sanku Mallik

e,

c,d,

Yongki Choi

Snigdha Banerjee

b,

c,d,

a*

Department of Coatings and Polymeric Materials, North Dakota

State University, Fargo ND 58108 b

Materials and Nanotechnology Program, Department of Physics,

North Dakota State University, Fargo ND 58108 c

d

Cancer Research Unit, VA Medical Center, Kansas City, MO 64128 Department of Pathology and Laboratory Medicine, University of

Kansas Medical Center, Kansas City, KS 66160 e

Department

of

Pharmaceutical

Sciences,

North

Dakota

State

University, Fargo ND 58108 *E-mail: [email protected]

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ABSTRACT: Hyperbranched polymer-derived drug nanocarriers have been synthesized that can change sizes selectively in response to pH. These constructs were composed of tertiary amine-conjugated polycarbonate blocks ‘grafted from’ a polyol

core.

At

neutral

pH,

hyperbranched polyester

unprotonated

polycarbonate

arms

stabilized the copolymer aggregates in the form of nanoparticles with hydrodynamic diameters ranging from 150-190 nm. Upon lowering of

pH,

these

larger

aggregates

disassembled

into

smaller

nanoparticles with diameters of 3-5 nm as directed by protonation of

tertiary

amine

side-chains.

The

pH-dependent

reduction

of

particle sizes was evident by titrimetric, spectroscopic, dynamic light

scattering,

transmission

electron,

and

atomic

force

microscopy-based experiments. We observed that these copolymeric nanoparticles could be loaded with dye and drug molecules either by non-covalent encapsulation or by covalent conjugation. A pHinduced disassembly of the aggregates initiated rapid release of the encapsulated payload, but not of the conjugated ones, thus establishing a controlled rate of therapeutic delivery from the nanostructures over an extended period. We envision that such systems

can

be

used

for

drug

delivery

applications

where

nanoparticle sizes critically govern therapeutic efficiency in a

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vasculature-poor disease microenvironment such as desmoplasia in pancreatic

cancer.

Hence,

we

tested

the

cellular

uptake,

cytotoxicity and chemotherapeutic potential of the size-modifiable nanoaggregates using gemcitabine as a model drug in pancreatic cancer setting. We observed that assembled nanoparticles were biocompatible to non-cancerous cells, showed pH-dependent effects on cellular uptake as well as promoted accumulation within cancer cells cultured as 3D spheroids. We also found that, when conjugated with

gemcitabine,

suppressed studies

the

proliferation

suggested

resulting of

that

cancer

these

drug-loaded cells.

nanoparticles

Collectively,

synthesized,

the

pH-disassembling

nanoscale platform will find applications as biomaterials for constructing

a

size-transformable

drug

nanocarriers

where

reduction of size takes effect near localized disease targets in response to microenvironmental triggers.

KEYWORDS: Size-modifiable nanoparticles, biomaterials, pHactivation, hyperbranched polymers, Chemotherapy Delivery INTRODUCTION One of the critical challenges in the field of biomaterials development is the ability to create synthetic macromolecules that is structurally stable, functionally programmable and capable of performing

therapeutically

desired

tasks

at

defined

physio-

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pathological

space

time.1-4

and

Page 4 of 81

Hyperbranched

polymers,

particularly those with terminal hydroxyl groups are one such attractive macromolecular platforms that have found traction in designing nanoscale architectures.5-8 Owing three-dimensional capabilities, functional components

structures,

the

groups such

as

presence and

intrinsic of

protein

molecular

many

inertness and

to their condensed confinement

modifiable,

peripheral

ubiquitous

biological

to

phospholipid

membranes,

these

highly branched polyols have established themselves as a novel molecular platform in biomaterials research, particularly in the area of drug delivery.9-21 For example, hyperbranched polyglycerols have been introduced by Haag et al.22-24 showed molecular transport of wide-spectrum of small molecular agents, while high molecular weight polyglycerols developed by Brooks et al.25, 27,

28

showed

accumulation

within

the

tumor

26

and by others

microenvironment,

mediated by enhanced permeation and retention (EPR) effect.29,

30

Hybrid structures, such as linear-dendritic polymers have also been

investigated

to

harness

the

capacity

fundamental to branched architectures.31,

of

multivalency

32

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Figure 1. Concept of pH-dissociable nanoparticles composed of drug conjugated hyperbranched architectures: (a) structure of hyperbranched polyester polyol (1) (b) Mechanism of pH-induced assembly-disassembly

of

the

nanoparticles

mediated

through

protonation/deprotonation of grafted segments. One of the major challenges for unimeric hyperbranched nanoconjugates involve short plasma residence time compared to their linear analogues and supramolecular assemblies due to rapid renal and hepatic clearance. We envision that if programmable nanoaggregates of hyperbranched polymers can maintain their sizes between 100-150 nm in plasma, and disassemble only at the disease microenvironment to its constituent branched unimers within 2-5 nm range (Figure 1), will increase the circulation lifetime of the aggregates while retaining the gain of multivalent advantage of hyperbranched polymers, i.e., high-affinity receptor interactions and enhanced membrane transport at the cellular level.

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Nanoparticles for delivery of chemotherapeutics needs to exhibit an optimum size range regarding hydrodynamic diameter for maximum therapeutic potential.33 While liver macrophages and Kupfer cells easily detect particles greater than 100 nm, those smaller than 5 nm are cleared out by renal filtration.33,

34

Hence, the proposed

constructs can bring in unique features of evading clearance mechanisms in systemic circulation, thus enhancing accumulation in tissue targets. Size-transformable nanocarriers are critically required for different cancer phenotypes, particularly those that are

characterized

by

dense

desmoplastic

stroma

and

altered

microvasculature, such as the cancer of the breast, testis, and pancreas. Poor and under-developed vasculature and thick collagen disposition are hallmarks of desmoplastic tumors, which often reduce the effective concentration of chemotherapy accumulating within

cancer microenvironment.

A programmable

drug delivery

system that breaks down to smaller nanoscale units in response to tumor microenvironment will also be beneficial for delivering chemotherapeutic agents into deep-seated neoplastic tissues that are devoid of fully matured blood vessels or exhibiting high solid stress.

We

were

motivated

to

translate

such

thermodynamic

assembly-disassembly behavior, particularly those governed by pH, into hyperbranched systems because one of the most prominent cellular heterogeneity observed in cancer is acute pH-drop within the

cancer

tissue,

which

increases

with

distance

from

blood

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ACS Biomaterials Science & Engineering

vessels.35 In addition, many cellular compartments, such as early endosome and lysosomes are inherently acidic compared to cytosolic pH.36,

37

In contrast to large cohort of nanoscale drug delivery

systems that utilizes the pH gradient of disease microenvironment as a trigger for initiating conformational change leading to drug release, we are using this pathological stimuli to transform nanocarrier

size

to

maximize

their

target

accumulation

and

penetration.38-40 To realize these constructs, we have harnessed the terminal hydroxyl groups of a hyperbranched polyester polyol (as shown in 1,

Figure

1a)

as

macroinitiators

to

initiate

ring-opening

polymerization of cyclic carbonates resulting in a grafted block copolymer.20,

41,

42

Post-functionalization

modification

of

the

resulting copolymer has been carried out to include pH-responsive tertiary amines along the grafted block.

Our central hypothesis

was

(ester)-b-linear

to

generate

a

hyperbranched

poly

poly

(carbonate) type graft copolymer, where the grafted arms will act as pH-sensitive domains that will induce aggregation at neutral and physiological pH and will promote disassembly into unimers at acidic pH mediated by protonation of side-chain amines (Figure 1b).43 We used this architecture in pancreatic cancer model because the heavy desmoplastic barrier of this type of tumor and altered microvasculature

hinders the diffusion of drug-loaded, larger

polymer assemblies (such as liposomes or polymersomes) into the

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cancer microenvironment. In addition, we loaded these copolymeric grafted

constructs

with

a

frontline

chemotherapeutic

agent,

gemcitabine, either by supramolecular encapsulation or by covalent conjugation to evaluate the general applicability of the system in pancreatic cancer.

EXPERIMENTAL SECTION Materials. All chemicals were obtained from Sigma-Aldrich, and anhydrous solvents were purchased from VWR, EMD Millipore.

1H

NMR

Spectra were recorded using a Bruker 400 MHz spectrometer using TMS as the internal standard. IR Spectra were recorded using an ATR

diamond

tip

on

a

Thermo

Scientific

Nicolet

8700

FTIR

instrument. Gel permeation chromatographic measurements were done on a GPC system (EcoSEC HLC-8320GPC, Tosoh Bioscience) using a differential RI detector, employing polystyrene (Agilent EasiVial PS-H 4mL) as the standard and THF as the eluent with a flow rate of 0.35 mL per minute at 40 °C. The sample concentration used was 1 mg/mL of which 20 µL was injected. DLS measurements were carried out using a Malvern instrument (Malvern ZS 90). UV-Visible and fluorescence

spectra

were

recorded

using

a

Varian

UV-Vis

spectrophotometer and a Fluoro-Log3 fluorescence spectrophotometer respectively. TEM studies were carried out using a JEOL JEM-2100

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ACS Biomaterials Science & Engineering

LaB6

transmission

electron

microscope

(JEOL

USA)

with

an

accelerating voltage of 200 keV. Material Synthesis. Hyperbranched poly (ester)-b-poly (carbonate) was synthesised first by using a preformed hyperbranched macroinitiator (hyperbranched bis-MPA polyester64-hydroxyl, generation 4, Mn=7323.32 g/mol, 1), through a ring opening polymerization of pentafluorophenol protected bis (methoxy propionic acid) derivative 2, following the procedure described by Hedricks et al.

20, 41, 42

(Scheme 1). Hyperbranched

Initiator (1, 13.7 µmol) was mixed with the monomer (cyclic carbonate, 2, 43.7 mmol and 87.4 mmol) to generate a set of

O O OH (1)

O

N

+ O F F

H N

N

O

F (2)

r.t., THF, 17h F F

O O

O O F

O

H

R-NH2, TEA

n

DMF, 24h ,r t.

O O

O O

F

F

F F

R = HN

3a + DBA = 3a-1 (3) 3a + PYR = 3a-2 3b + DBA = 3b-1 3b + DBA + Gem (covalent) = 3b-cn 3b-1 + Gem (non-covalent) = 3b-en

NH R

H n

n = 119, 3a n = 240, 3b

NH

HN

N

or N

(DBA)

N (PYR)

HO

O F OH

F

N

O

Gemcitabine

Scheme 1. Synthetic route for hyperbranched pH-responsive block copolymers.

compound 3a and 3b, respectively with different lengths of the grafted block. 1, 5, 7-Triazabicyclo [4.4.0] dec-5-ene (TBD, 0.014 mmol) was used as the catalyst for this polymerization.

1H

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NMR of 3a is presented in Figure S1 (Supporting information). The pentafluorophenyl ester (PFP-ester) groups of polymer 3a were replaced using two different types of tertiary amines, i.e. N, Nʹdibutylethylenediamine (DBA, pKa = 4.0), or 2-pyrrolidin-1yl-ethyl-amine (Pyr, pka = 5.4) using the procedure mentioned by Engler et al.

41

with slight modifications. To synthesize amine-

conjugated copolymers, the polyester-b-poly (carbonate) with PFP ester (3a, 1.0 µmol) was dissolved in DMF and cooled in an ice bath. A 0.5 mL DMF solution of corresponding amines (0.58 mmol or 0.88 mmol for DBA and Pyr, respectively) was added dropwise to the polymer solution with constant stirring for synthesizing 3a-1 or 3a-2, respectively. Triethylamine was used as the catalyst for this reaction. The solution was left to stir for 24 hours at room temperature followed by precipitation in cold diethyl ether. This general procedure generated a set of pHresponsive graft copolymers, 3a-1 and 3a-2, respectively where 1 and 2 indicates the N, Nʹ-dibutylethylenediamine and 2pyrrolidin-1-yl-ethyl-amine substituted derivatives of the corresponding block copolymers. Compound 3b-1 was further synthesized for testing non-covalent encapsulation of gemcitabine. Overall yields for the amine-substituted polymers were typically found to be around 50-60% depending on the amine substitution and solubility in precipitating solvent. The synthesized products were characterized using

1H

NMR and IR

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ACS Biomaterials Science & Engineering

spectroscopy.

1H

NMR of 3a-1 (DMSO-d6): δ 0.9 (t, 3H, -N-CH2-CH2-

CH2-CH3), 1.3 (m, 2H, -N-CH2-CH2-CH2-CH3), 1.4 (m, 2H, -N-CH2-CH2CH2-CH3), 2.4 (m, 2H, N-CH2-CH2-CH2-CH3), 2.45 (2H, m, -NH-CH2-CH2N(C4H9)2), 3.0 (m, 2H, -NH-CH2-CH2-N(C4H9)2), and (DMSO-d6):

1H

NMR of 3a-2

δ 1.7 (m, 2H, -N-CH2-CH2-CH2-CH2-), 2.7 (t, 2H, -N-

CH2-CH2-CH2-CH2-), 2.48 (m, 2H, NH-CH2-CH2-N(C4H8)2), 3.1 (m, 2H, NH-CH2-CH2-N-(C4H8)2 (Figure S1, b-c, Supporting Information). Molecular weights of the copolymers bearing PFP ester were determined by gel permeation chromatography using THF as eluent. Number average molecular weight (Mn) of compound 3a and 3b was found to be 42.9 kDa and 79.0 kDa respectively (Figure S2a for GPC trace of 3a, Supporting Information). Complete replacement of the esters in these parent macromolecules by respective amines was further confirmed by

19F

NMR that showed no fluorine

signals in the aminated compounds (Fig. S2b for 3a-1, Supporting Information).

IR spectroscopy showed the appearance of the peak

corresponding to the amide stretching frequency at 1662 cm-1 for the aminated polymers (Figure S2c for 3a-1, Supporting Information). Synthesis of drug conjugated polymers. Compound 3b was selected for drug encapsulation or conjugation due to its physicochemical properties (i.e. molecular weight and solubility) optimum for drug loading. We either encapsulated gemcitabine

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inside nanoparticles formed from 3b-1, yielding 3b-en (encapsulated) systems or co-conjugated to 3b in the presence of the drug and DBA yielding 3b-cn (conjugated) constructs. For preparation of 3b-cn systems, compound 3b (0.6 µmol) was reacted with gemcitabine (17 µmol) and N, Nʹdibutylethylenediamine (260 µmol) following the amination procedure mentioned in the previous section (Figure S3, Supporting Information). The characteristic gemcitabine signals were observed in the spectrum of 3b-cn constructs at δ 6.1 (d, 1H,

1H

NMR

-NH-CH=CH-N-C),

8.0 (d, 1H, -NH-CH=CH-N-C), 4.1 (br, m, 1H, -OH-CH2- CH (O)´), 3.6 - 3.8 (m, 2H, -OH-CH2-CH (O) R´). Identification codes of all compounds and nanoparticle systems synthesized are presented in Scheme 1. Quantification of gemcitabine Payload in drug conjugated polymer: The drug extraction from the polymer-drug conjugate (3b-cn) was performed by subjecting the conjugate to alkaline hydrolysis using 1N NaOH for 1h at 40°C as reported by Mahato et al.44 After hydrolysis, the mixture was centrifuged for 30 minutes at 10,000 rpm using ultra centrifuge filters (MWCO 3k). The filtrate was analyzed using UV-Visible spectroscopy and the payload was calculated using equation (1) .44: 𝑤 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑒𝑑 = × 100 𝑤 𝑇𝑜𝑡𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑓𝑜𝑟𝑚𝑢𝑙𝑎𝑡𝑖𝑜𝑛

𝑃𝑎𝑦𝑙𝑜𝑎𝑑(%)

(1)

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For gemcitabine encapsulated in nanoparticles, the following formula was used to calculate encapsulation efficiency (Equation 2)

45:

𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑒𝑛𝑐𝑎𝑝𝑠𝑢𝑙𝑎𝑡𝑖𝑜𝑛 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦(%) = 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑎𝑑𝑑𝑒𝑑 ― 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑒𝑛𝑐𝑎𝑝𝑠𝑢𝑙𝑎𝑡𝑒𝑑 × 100 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑎𝑑𝑑𝑒𝑑

(2)

Determination of pKa: Acid dissociation constant, i.e., pKa values of the synthesized polymers and the corresponding monomers were determined by titrating the aqueous solution (5.0 mL) of these polymers against 0.1 N sodium hydroxide. Determination of the Critical Aggregation Concentration (CAC) of the polymers: To evaluate the aggregate forming capacity of the copolymers and to estimate their systemic stability, we determined the Critical Aggregation Concentration (CAC) of the synthesized architectures. A stock solution of 0.1 mM pyrene in dichloromethane was prepared. An aliquot of 10 µL of this solution was taken in a set of vials, and dichloromethane was allowed to evaporate by air-drying. To each of these vials, various measured amounts of the aminated polymers were added (from a stock solution of 1 mM) so that the concentrations varied from 1.9 μM to 0.5 mM and the final concentration of

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pyrene in each vial remained 1 µM. The vials were sonicated for 1 hour 30 minutes and then allowed to stand for 3 hours before recording the fluorescence spectra.46 The fluorescence emission spectra were acquired at an excitation wavelength of 337 nm with slits of 2.5 nm (for both excitation and emission). The ratio of the intensities at 373 nm and 384 nm were plotted against the concentration of the polymer, and the inflection point of the curve was used to determine the CAC. Preparation

of

Nanoparticles:

To

prepare

self

assembled

structures from the grafted block copolymers, nanoprecipitation or solvent shifting method from a selective solvent (DMSO) to a nonselective

solvent

(buffer)

was

employed.

Copolymers

such

as

compound 3a-1 and 3a-2 (10 mg) were dissolved in 250 µL of DMSO, and the solution was added drop-wise to 750 µL of PBS buffer (pH 7.4). The resultant solution was transferred to a Float-a-lyzer® (MWCO 3.5-5 kDa) and dialyzed against 700 mL PBS buffer (pH 7.4) overnight with constant shaking at moderate speed. For gemcitabine encapsulated nanoparticle systems (3b-en), 10mg of the polymer (3b-1) and 5 mg gemcitabine were dissolved in 250 µL DMSO and added dropwise to a 750 µL of PBS (pH 7.4) with constant stirring. The solution was allowed to stir overnight followed by filtration using an ultracentrifuge filter (MWCO 3.5-5 kDa) at 5000 rpm for 3 hours to

prepare

the

purified

nanoparticles.

Then

the

resulting

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nanoparticle suspension was redispersed in chilled (4⁰C) PBS to a concentration of 10 mg/mL. The filtrate was used to quantify the amount of drug. For preparation of nanoparticles with gemcitabine conjugated copolymer (3b-cn), 10 mg of 3b-cn were dissolved in 250 µL DMSO and added dropwise to 750 µL of a stirring solution of PBS (pH

7.4),

and

procedure

similar

for

constructing

3b-en

nanoparticles were followed. Particle size and Zeta Potential analysis of nanoparticles: The hydrodynamic diameters of resulting nanoparticles prepared from copolymers 3a-1 and 3a-2 were determined using Dynamic Light Scattering (DLS) at a scattering angle of 90°. For zeta potential measurements a sample concentration of 10 mg/mL was used and the zeta potential was determined in terms of electrophoretic mobility by taking an average of 5 readings. For all of these measurements, the sample solution was filtered using a 0.2 µm PES filters. Release of nanoparticle-encapsulated model compound: We used Alexa Fluor-647 as a pH-independent model compound to investigate non-covalent encapsulation and pH-mediated content release from resulting nanoparticles. We have chosen this dye due to its pH insensitivity in the range of pH 4-10. The dye encapsulation was carried out by preparing a stock solution of 1 mg/mL of Alexa Fluor 647 in DMF and adding 50 µL of the dye solution to 250 µL respective copolymer (3a-1, 10 mg/ mL). The resulting solution was added to 700 µL of PBS in a drop-wise pattern with constant stirring

45.

The

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Page 16 of 81

resulting solution was allowed to stir for an hour and dialyzed overnight using a Float-a-lyzer® (MWCO 3.5-5 k Da) against 800 mL PBS buffer. The media was changed, and dialysis continued for another 6 hours until no further discoloration of the media was observed in order to ensure complete encapsulation of the dye by nascent

nanoparticles.

The

release

of

the

dye

from

these

nanoparticles was studied at different pH (i.e., 4.5, 5.5, and 7.4). Starting from pH 7.4, the pH of the media was reduced every 3 hours until the end of 9 hours. To evaluate the amount of dye released, the fluorescence emission intensity of the sample was recorded at 655 nm by excitation at 647 nm.

After 9 hours, 20 µL

of Triton was added to disintegrate the nanoparticles and the fluorescence emission intensity was measured for total release after

disintegration.

The

cumulative

percentage

release

was

plotted versus elapsed time. Percentage release was calculated using the following equation: % 𝑟𝑒𝑙𝑒𝑎𝑠𝑒 =

𝐸𝑚𝑖𝑠𝑠𝑖𝑜𝑛 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑎𝑓𝑡𝑒𝑟 𝑟𝑒𝑙𝑒𝑎𝑠𝑒 ― 𝐸𝑚𝑖𝑠𝑠𝑖𝑜𝑛 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑏𝑒𝑓𝑜𝑟𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒 𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑎𝑓𝑡𝑒𝑟 𝑇𝑟𝑖𝑡𝑜𝑛 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 ― 𝐸𝑚𝑖𝑠𝑠𝑖𝑜𝑛 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑏𝑒𝑓𝑜𝑟𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒

× 100

(3)

Size analysis using TEM imaging: A drop of nanoparticle sample (obtained from copolymer 3a-1) was placed on a 300-mesh formvarcarbon coated copper TEM grid (Electron Microscopy Sciences) for 1 min and wicked off.

Phosphotungstic acid 0.1%, pH adjusted to

7-8, was dropped onto the grid and allowed to stand for 2 minutes and then wicked off. Nanoparticles incubated both at pH 7.4 and

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4.5 has been investigated for their microstructure by TEM at 200 KeV. pH-dependent dissociation of nanoparticles: The disassembly of the nanoparticles prepared from amine substituted poly (ester)-gpoly

(carbonate)

was

studied

by

the

probe-probe

quenching-

dequenching method described by Kataoka et al.47 The self-assembled structures used for this study were prepared by covalent attachment of

Alexa

Fluor

647

dye

to

the

copolymer,

3a-1,

followed

by

nanoprecipitation. Emission intensity of the nanoparticle solution was measured at 665 nm by exciting the sample at 650 nm, incubated at two different values of pH (4.5 and 7.4) for a stipulated period. The emission intensity was plotted versus time to monitor the evolution of fluorescent signal due to dequenching of the dye over elapsed time. Atomic Force Microscopy of pH-dissociable nanoparticles: The nanoparticle samples derived from 3a-1 were incubated in two different buffer solutions (10 mM, pH 7.4 and pH 4.5). The imaging samples were prepared by incubating 10 L of each solution on silicon substrates for 30 min in a sealed compartment to protect evaporation at room temperature. The samples were then rinsed with de-ionized water (Millipore), and dried under N2 flow. The imaging measurements

were

performed

using

a

commercial

atomic

force

microscope (NT-MDT NTEGRA AFM). The samples were imaged under

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Page 18 of 81

ambient conditions in semi-contact mode with a resonant frequency of 190 kHz AFM probes (Budget sensors). In Vitro release of gemcitabine: In vitro drug release was studied at two different conditions at pH 7.4 (PBS buffer) and 4.5 (PBS buffer lowered by adding 0.1 M HCl). An aliquot of 1 mL of the drug-conjugated nanoparticles (formed from copolymer 3b-cn) and encapsulated nanoparticles (from 3-en) was taken in different Float-a-lyzer® (MWCO 3.5-5kDa) chambers against 5 mL of media. After a specified time interval, 1 mL of sample was withdrawn and replaced with the same volume of fresh media. The samples were then

analyzed

for

gemcitabine

concentration

using

UV-Vis

spectroscopy. Cell

viability

assay.

Cytotoxicity

of

gemcitabine-conjugated

nanoparticles from copolymer 3b-en was tested on pancreatic cancer cells, MiaPaCa-2. For this experiment, 1000 cells/well were seeded in 96-well plates and 24 h later, were treated with different concentration of drug conjugated nanoparticles and the equivalent concentration of free gemcitabine. After incubating the cells for 72 hours, cell viability assay was performed by MTS assay. Cell viability was calculated using equation (4): 𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) =

𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡𝑒𝑠𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 × 100 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙

(4)

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Cellular uptake studies by flow cytometry. MiaPaCa-2 cells were plated in 6-well plates at 5,000 cells/well and were allowed to grow to 70% confluence. The cells were then incubated at 37 ⁰C overnight with dye (Alexa Fluor 647) labelled nanoparticles assembled from copolymer 3a-1. After the incubation period, cells were washed with cold PBS (3X), trypsinized, and resuspended in PBS. Subsequently, the cells were suspended in a flow cytometry buffer (PBS with 0.1% BSA), and the cellassociated fluorescence intensity of Alexa Fluor 647 was determined by BD Biosciences Accuri C6 Flow Cytometer. Confocal

microscopy

of

monolayer

and

spheroid

culture

of

pancreatic cancer cells. To evaluate the pH-dependent uptake of dye-conjugated nanoparticles, Panc-1 pancreatic cancer cell lines were used. Panc-1 cells were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St Louis, MO) supplemented with 10% Foetal bovine serum (ATCC, Manassas, VA) and 10% glucose. After trypsinization, cells were seeded on a glass cover slide at a concentration of 104 cells, and were incubated overnight with dyelabelled nanoparticles (formed from 3a-1) at pH of 7.4 and 4.5 at a concentration of 10μg/ml. After 24 h, the cells several

times

in

PBS,

fixed

with

4%

were washed

paraformaldehyde

and

permeabilized with 0.1% TritonX-100. Cells were counterstained with

nuclear

stain DAPI and

were imaged

using

a

confocal

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microscope (Leica, Wetzlar, Germany). To determine the capacity of nanoparticles to penetrate pancreatic cancer cells clustered in 3D, spheroid cultures of MiaPaCa-2 cells were used. Such models are used to mimic the 3D-native environment of pancreatic cancer lacking blood vessels. Spheroid cultures have been reported to recapitulate

hypoxic

and

acidic

conditions

of

desmoplastic

tumors48-54. To grow these cultures, cells growing in culture flasks were

treated with the

NanoShuttleTM (n3D

Biosciences courtesy

Greiner Bio) solution overnight, following which the cells were washed, trypsinized and seeded in a 96 well plate with the cellrepellent surface (n3D Biosciences courtesy Greiner Bio). Seeding density was maintained at 1 x 106 cells per spheroid, and the plate was incubated on the spheroid drive (n3D Biosciences, Greiner Bio) in the incubator overnight. After 24 hour of incubation and when the spheroids were visible, they were treated with dye labelled nanoparticles (100 μL at 10 mg/mL concentration, formed from copolymer 3a-1) and incubated for another 24 hours. Following this incubation period, the spheroids were washed with PBS (while placed on a holding drive so as not to disrupt the spheroids), stained with DAPI and analyzed using confocal microscope.

RESULTS AND DISCUSSION

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Synthesis and characterization of pH-responsive graft

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copolymers: The pH-responsive block copolymers, i.e. compounds

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3a-1 and 3a-2, and their drug loaded analogues, i.e. 3b-en and

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3b-cn were synthesized from the copolymer 3a and 3b

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respectively, which were realized by ring-opening polymerization

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of pentafluorophenyl ester appended activated carbonate monomer

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Page 27 of 81

(2), initiated by a hyperbranched polyester polyol (1) of generation 4. As observed from Figure 2a, for all variants of compound 3, protons corresponding to the –CH2-O-CO- in the graft copolymer were located at δ 4.1-4.6 ppm, and the -CH2-C-CH2- of the hydrophobic arms were observed at δ 4.3 and 4.5 ppm

(a)

(b)

(c)

(d) 1.55

pH 7.4 pH 4.5

1.5 1.45

I1/I3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

1.4

1.35 1.3 0

Figure 2. (a)

1H

0.005 0.01 0.015 0.02 Polymer concentration (mM)

NMR spectra of compounds 1 (hyperbranched

polyester polyol), 3a and 3a-1 showing the appearance of the CH2 signals in 3a and the appearance of the methyl group from the dibutyl chains of the amine in 3a-1 as indicated by the arrows (b) pH-titration curve of compounds 3a-1 and 3a-2 and their respective monomers (c) Pyrene fluorescence spectra in the 19F NMR (detailed S1,Ratio Supporting presence in of Figure 3a-1 (d) of I1/IInformation as a3a). function of 3 of pyrene for

concentration of 3a-1, in two different pH conditions (i.e. pH 7.4 and 5.0).

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spectra with trifluoroacetic acid as internal standard revealed the appearance of fluorine signal at -160 ppm from pentafluorophenyl ester in compound 3a (Figure S2b, Supporting Information). The molecular weight of compounds 3a-b was analyzed using gel permeation chromatography, using THF as an eluent.

The number average molecular weight of compound 3a was

found to be 42.9 kDa (Figure S2a, Supporting Information), and 79 kDa was found for compound 3b. The GPC data showed an increment of molecular weight of the hyperbranched initiator (Mn=7323.32 g/mol) by 35,576 and 71,677 g/mol was obtainable with this polymerization method, indicating the inclusion of at least 119 and 240 units of PFP ester units appended to carbonate monomer on the hyperbranched initiator for compounds 3a and 3b respectively. When compound 3a was reacted with tertiary amines, the IR spectra of the resulting compound 3a-1 and 3a-2 showed the characteristic peak at 1662 cm-1 for stretching vibrations of the amide bond (Figure S2c, Supporting Information) with complete disappearance of fluorine signals in

19F

NMR (Figure

S2b, Supporting Information) indicating complete functionalization. We also showed that these synthetic platforms could also be used conveniently to generate drug-conjugated block copolymers. To this end, compound 3b was covalently linked with gemcitabine using similar transamination reaction as adopted for connecting tertiary amines.44 For such conjugation,

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compound 3b was treated with a stoichiometric mixture of the corresponding amine and the drug, resulting in the formation of pH-responsive graft copolymer where a specific quantity of gemcitabine was attached to the side chain of the grafted arm in the resulting product, 3b-cn (shown in Scheme 1 earlier). The amount of attached gemcitabine was later quantified. pH-responsivity and self-assembly of assembled nanoparticles: Tertiary amines are well-established pH-responsive motifs, which we have introduced in the grafted arm of these copolymers to regulate the pH-triggered association-disassociation behaviour of the assembled nanoparticles. Figure 2b shows the titration curve of compound 3a-1 and 3a-2 from which the net pKa value (pH at half inflection)

of

the

amine-appended

block

copolymers

can

be

estimated. The calculated pKa value for compound 3a-1 was found to be 6.8 (±0.23, n=3), which led us to infer that above this particular pH, most of the grafted arm of the copolymer will be unprotonated, leading to the formation of nanoparticles stabilized by hydrophobic interactions operated through the unionized grafted blocks emanating from the core. At or below pH 6.8, at least 50% of the amines located along the grafted segment will be protonated. Such ionization will trigger the hydrophilic phase transition and disruption of self-assembled structures to form smaller aggregates and unimers. To test this hypothesis, we have investigated the

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Page 30 of 81

reversibility of the system, i.e. a reverse titration was performed using 0.1 N HCl, and the graft copolymers were found to be fully reversible

in

their

protonation-deprotonation

behavior.

While

titrating to a basic condition by incremental addition of NaOH, we found that the particles precipitated out of the solution at pH 10, forming a turbid solution. However, this precipitation and turbidity were no longer observed when the reverse titration was carried out, indicating that the protonation of side chain amines might be responsible for the disruption of the aggregates at lower pH.

For compound 3a-2, the pKa value was found to be 7.08 (± 0.15,

n=3, Figure 2b). To investigate if such hyperbranched systems can form self-assembled structure at the molecular scale, we set out to conduct fluorescence spectroscopic experiments using pyrene to determine the critical aggregation concentration (CAC) of the graft copolymers.46 The first (λ373 nm) and third (λ384 nm) peaks in the fluorescence emission spectra of pyrene are sensitive to the polarity of the environment and, the ratio of the intensities provides information regarding the stability of association of the polymers.55 When tested with compound 3a-1, we observed that the ratio of I373/I384 decreased with increasing copolymer concentration after which it remained almost unchanged (Figure 2c). The value of I1/I3

at the

point

of

intersection (Figure 2d) leads to

the

inference that the probe is located in a hydrophobic environment of 3a-1 nanoassembly, created by the aggregation of the hydrophobic

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segments above the CAC of this grafted copolymer. The CAC value of 3a-1 was determined to be 1.29 x 10-6 M and that for 3a-2 as 5.16 x 10-6 M.

Lower CAC values for the N, Nʹ-dibutylethylenediamine

containing system can be attributed to the alkyl chains of the tertiary

amines,

entanglement

than

which the

tend

to

alicyclic,

form

stronger

compact

hydrophobic

structure

of

the

pyrrolidone amines in 3a-2. To observe the disassembling effect of acidic pH on the formation of aggregates, the same experiment was carried out at pH 4.5, and no clear break point was observed at this pH. Instead, we have monitored a gradual decrease in the ratio of the intensities with an increase in polymer concentrations (Figure 2d). This leads to the inference that at an acidic pH these block copolymers do not self-assemble to stable, larger aggregates due to intermolecular electrostatic repulsion originating from the protonation of tertiary amine side chains on the grafted block.

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The synthesized block copolymers of 3a-1 and 3a-2 were found to form

self-assembled

nanoparticles

under

nanoprecipitation

condition from DMSO to an aqueous buffered solution of pH 7.4. While

3a-1

formed

nanoparticles

with

an

average

hydrodynamic

diameter within the range of 182 ± 15.4 nm (n=5) (Figure 3a), nanoparticles composed of 3a-2 exhibited an average diameter of

Figure

3:

(a)

Dynamic

light

scattering

of

nanoparticles

resulting from aggregation of 3a-1, showing larger particles dominate at pH 7.4, which converts to smaller particles when the

pH

is

lowered

nanoparticles gradual

to

which

reduction

of

4.5

(b)

dissociate pH,

and

AFM to (c)

images smaller

showing particles

Organized

intact upon

nanostructure

(leftmost panel) of 3a-1 nanoparticles, which increases in ACS Paragon Plus Environment dispersity and decreases in size as pH is gradually lowered to 32 pH 4.5.

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153 ± 16.9 nm (n=5) when formed under similar conditions. These nanoparticle solutions were found to be stable at least for up to 6 weeks when stored at 4°C at pH 7.4 (Figure S4a, supporting Information). At pH 4.5, however, these nanoparticles showed a reduction in size. For example, when nanoparticles derived from copolymer 3a-1, were incubated at pH 4.5 and the particle sizes were measured, no larger aggregates were evident, somewhat smaller diameter-containing species were observed with an average diameter of 2.95 ± 3.1 nm (n=3). This observation is possibly due to the breakdown of the larger aggregates into hyperbranched copolymeric unimers (Figure 3a). We observed that such reduction of number average particle size took place in a pH dependent manner via intermediate stages of pH 6.5 (124.8 ± 8.97 nm) and pH 5.5 (54.76 ±7.02 nm). Atomic force microscopic (AFM) observations further corroborated our hypothesis. As evident in Figure 3b (at pH 7.4 in AFM image panel), nanoparticles derived from 3a-1 existed as nanostructures with well-defined, circular particles with a mean diameter

of

~

100

nm

at

pH

7.4

but

showed

smaller,

multi-

particulate structures (