In vitro controlled release of tuberculosis drugs by amphiphilic branched copolymer nanoparticles

Accepted Manuscript Title: In Vitro Controlled Release of Tuberculosis Drugs by Amphiphilic Branched Copolymer Nanoparticles Authors: Mani Gajendiran, Heejung Jo, Kyobum Kim, Sengottuvelan Balasubramanian PII: DOI: Reference:

S1226-086X(19)30194-7 https://doi.org/10.1016/j.jiec.2019.04.033 JIEC 4513

To appear in: Received date: Revised date: Accepted date:

28 November 2018 19 April 2019 20 April 2019

Please cite this article as: Gajendiran M, Jo H, Kim K, Balasubramanian S, In Vitro Controlled Release of Tuberculosis Drugs by Amphiphilic Branched Copolymer Nanoparticles, Journal of Industrial and Engineering Chemistry (2019), https://doi.org/10.1016/j.jiec.2019.04.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

In Vitro Controlled Release of Tuberculosis Drugs by Amphiphilic Branched Copolymer Nanoparticles

Mani Gajendiran,a,b, Heejung Jo,b, Kyobum Kim,b,*, Sengottuvelan

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Balasubramaniana,*

a

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Department of Inorganic Chemistry, University of Madras, Guindy Campus,

Chennai-600025, India.

Division of Bioengineering, School of Life Sciences and Bioengineering,

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b

Corresponding author: Prof. Dr. Sengottuvelan Balasubramanian,

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*

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Incheon National University, Incheon-22012, Republic of Korea.

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Department of Inorganic Chemistry, University of Madras, Guindy Campus, Chennai-600025, India., E-mail: [email protected]

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Prof. Dr. Kyobum Kim, Division of Bioengineering, School of Life Sciences

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and Bioengineering, Incheon National University, Incheon-22012, Republic of

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Korea., E-mail: [email protected]

Graphical abstract A series of Citrate-PEG-PLGA branched amphiphilic copolymer nanoparticles have been synthesized for tuberculosis drug delivery.

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Abstract

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Poly(lactic-co-glycolic acid) (PLGA)-poly ethylene glycol (PEG) based

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amphiphilic branched copolymer nanoparticles (NPs) have been developed for

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controlled release of tuberculosis (TB) drugs which include rifampicin (RIF),

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isoniazid (INH) and pyrazinamide (PYZ). The drug loading efficiency and the

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percentage drug content of polymer NPs increase by increasing the amount of PEG content in polymer NPs. The branched PLGA-PEG based copolymer NPs

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exhibit initial burst release followed by sustained release of RIF for 840 h, INH

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for 72 h, and PYZ for 720 h. The branched citrate-PEG-PLGA copolymer NPs

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can act as potential drug carriers when compared to their linear analogues.

Keywords: Citrate-PEG-PLGA branched copolymer, Polymer nanoparticles, In vitro drug release, Rifampicin, Isoniazid, Pyrazinamide,

Introduction 2

World health organization (WHO) in 2017 has declared that tuberculosis is ninth leading cause of death worldwide and indicated that 1.3 million tuberculosis (TB) deaths occurred among HIV negative people and an additional 0.374 million TB deaths were estimated among HIV-positive people

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in the year 2016. The TB is caused by mycobacterium tuberculosis and treated by long-term chemotherapy with the first line TB medicines rifampicin (RIF),

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isoniazid (INH), pyrazinamide (PYZ) and Ethambutol (ETB) for 9 to 12

months. Since, the long-term chemotherapy with high dose of first line TB

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drugs would result in multi-drug resistant TB (MDR), the second line TB drugs

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are used to treat MDR. Recently new drugs such as Bedaquiline and Delamanid

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are administered along with other TB medicines to treat MDR-TB [1, 2].

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However, the treatment of TB is complicated due to poor patient compliance.

Several polymer nanoparticles (NPs) based controlled drug delivery

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systems (CDDS) have been developed to minimize side effects and dosing

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frequency of the TB drugs, which provide a good therapeutic approach to improve patient compliance [3]. Various biocompatible polymers based CDDS

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have been developed for the sustained release of TB drugs [4]. For instance, Thomas et al. have reported alginate-cellulose NPs for the in vitro release of RIF, while Prabhakar et al. have employed chitosan-graftpoly(caprolactone)/(ferulic acid) nanomicelles for sustained release of RIF[5, 6]. Gelatin cellulose-whiskers NPs can be used for controlled release of INH 3

[7]. However, most of those polymers were either non-biodegradable or showed faster drug release. The poly (lactic-co-glycolic) acid (PLGA) based CDDS have been employed extensively for the controlled release of tuberculosis drugs due to their biocompatibility, biodegradability and ability to release TB drugs

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for a prolonged period under physiological conditions [8, 9]. However, most of the strongly entrapped drug molecules were not released from PLGA particles

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for a long period [10-12]. Since, PLGA is a hydrophobic polyester, they showed

very low loading efficiency for hydrophilic TB drugs such as INH and PYZ [13,

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14]. The PLGA-polyethylene glycol (PEG) based block copolymers acted as

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of their amphiphilic nature [9, 11, 15].

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prominent drug carriers for both hydrophilic and hydrophobic TB drugs because

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The PLGA-PEG based linear multiblock copolymers were synthesized by using dicarboxylates such as succinate and tartrate as linear linking agents and

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the obtained PLGA-PEG based linear multiblock copolymers were successfully

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used as potential drug carriers for controlled release of first line TB drugs including INH, RIF and PYZ [12, 16]. Recently, branched polymers have been

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developed for drug delivery applications which exhibit unique topological structures, interesting physico-chemical properties, greater encapsulation efficiencies and multi-functionality when compared to their linear analogues [17]. In recent years, several patents on branched polymers for various applications have been filed because of their versatility [18, 19]. Hence, the 4

PLGA-PEG based branched polymers can act as potential drug carriers for the controlled release of TB drugs.

In the present work, citrate-PEG-PLGA branched block copolymers are

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reported for the first time. The citric acid (CA) was reacted with PEG to obtain citrate-PEG (CAP) branched copolymer and then PLGA (79:21) was reacted

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with different weight ratio of CAP via direct melt condensation reaction to get a series of citrate-PEG-PLGA (PCP series) amphiphilic branched copolymers.

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The copolymers have been characterized by Fourier transform infrared

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spectroscopy (FTIR), proton magnetic resonance spectroscopy (1H-NMR), gel

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permeation chromatography (GPC) and inherent viscosity [η]. The tuberculosis

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drug RIF loaded PCP series polymer NPs were prepared by water-in-oil (W/O)

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single emulsion technique, while INH or PYZ loaded PCP series branched polymer NPs were obtained by water-in-oil-in-water (W/O/W) double emulsion

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technique. The in vitro drug releasing properties and drug loading efficiencies

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of the TB drug loaded PCP series amphiphilic branched copolymer NPs were compared with that of drug loaded hydrophobic PLGA microparticles and other

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formulations reported in literature.

Experimental section Materials

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DL-lactic acid (LA), glycolic acid (GA), citric acid (CA) (Merck specialties Pvt. Ltd., India), stannous chloride dihydrate (Fisher scientific, India), PEG6000 (HiMedia, India), isoniazid, rifampicin, pyrazinamide and poly vinyl alcohol 14000 (PVA) (Sigma–Aldrich) were used as received. All the solvents used

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were of analytical grade.

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Characterization methods

The inherent viscosity of polymer samples was measured by using an

H-NMR spectral analysis was performed on a Bruker 300 MHz NMR

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Ubbelohde viscometer using chloroform at a concentration of 0.1 g/dL at 40 °C.

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spectrometer in CDCl3. FTIR spectra were recorded on a Perkin-Elmer 8300

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FTIR spectrometer. UV–Visible absorption spectral analyses were carried on a

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Perkin-Elmer Lambda-35 UV–Visible spectrophotometer. FESEM images of drug loaded polymer NPs were captured on a HITACHI SU6600 field emission

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scanning electron microscope. TEM analysis was performed on a FEI TECNAI

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G2 (T-30) transmission electron microscope.

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Synthesis of PLGA The PLGA was synthesized by direct melt polycondensation of DL-LA

(0.36 mol; 32.42 g) and GA (0.0737 mol; 5.61 g) by esterification mechanism initiated by stannous chloride dihydrate as given in the literature [12, 20]. (Yield: 28.32 g ; 70 %). (mp = 102 oC; FTIR: 3500 cm-1, 3004 cm-1, 2948 cm-1, 6

2893 cm-1, 1759 cm-1, 1455 cm-1, 1190 cm-1, 1098 cm-1 and 756 cm-1; 1H-NMR ̅̅̅̅̅ = (CDCl3): δ5.2(q), δ4.6-4.9, δ1.5(d); [η]=0.14 dL/g; ̅̅̅̅ Mn = 1987 g/mol, Mw 2698 g/mol, PDI = 1.3).

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Synthesis of CA-PEG copolymer (CAP) CAP was synthesized by direct melt polycondensation with a

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modification of literature procedure [21]. A round bottom (RB) flask containing a mixture of 1 g of citric acid (CA), 10 g of PEG and 0.11 g of stannous chloride dihydrate was degassed for 30 min. Then it was heated to 160 oC for 2

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h under 15-25 mm Hg vacuum, and then it was cooled to room temperature. The

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product was dissolved in chloroform and precipitated in diethyl ether. Finally, it

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was washed thrice with diethyl ether and dried under vacuum overnight. (Yield:

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8.3 g ; 75.4 %). (mp = 40-43 oC; FTIR: 3450 cm-1, 2882 cm-1, 1733 cm-1, 1458 cm-1, 1192 cm-1, 1096 cm-1 and 758 cm-1; 1H-NMR(CDCl3): δ2.8 (s), δ3.65 (s);

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̅̅̅̅̅ = 3573 g/mol, PDI = 2.2). [η]=0.17 dL/g; ̅̅̅̅ Mn = 1588 g/mol, Mw

General method for the synthesis of a series of citrate-PEG-PLGA three arm

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block copolymers (PCP series) The PLGA (4 g), CAP (0.4, 0.8, 1.2 or 1.6 g) and stannous chloride

dihydrate (0.1 wt. %) were taken in a RB flask and degassed for 30 min. The

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reaction was carried out at 160 oC for 10 h under nitrogen atmosphere. Then the reaction mixture was cooled to room temperature, the resulting product was dissolved in chloroform and precipitated in diethyl ether. Finally, the product was washed thrice with diethyl ether and dried under vacuum. (Yield: 2.87 3.53 g ; 50 - 75 %). (mp = 48-66 oC; FTIR: 3500 cm-1, 3004 cm-1, 2948 cm-1, 7

2893 cm-1, 1760 cm-1, 1455 cm-1, 1190 cm-1, 1098 cm-1 and 756 cm-1; 1HNMR(CDCl3): δ2.2(s), δ5.2(q), δ4.6-4.9, δ1.5(d); [η]=0.18-0.24 dL/g; ̅̅̅̅ Mn = ̅̅̅̅̅ = 3573-5677 g/mol, PDI = 1.3-6.0). 735-4110 g/mol, Mw

Preparation of RIF loaded copolymer NPs

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RIF loaded polymer NPs were prepared by ultra-sonication-initiated W/O

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emulsion technique with a slight modification of literature procedure [11]. The PCP series copolymers (400 mg) and RIF (100 mg) were dissolved in 5 mL of dichloromethane (DCM), and then 5 mL of aqueous PVA (0.5 % (w/v)) was

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added under ultrasonication for 10 min using a probe-ultrasonicator (Hielscher-

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UP100H). Then it was stirred magnetically for 20 min. Double distilled (DD)

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water (10 mL) was added to the W/O emulsion and it was stirred for 60 min. The RIF loaded polymer NPs dispersion was initially centrifuged at 4612 RCF

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(×g) to remove the bigger sized particles and again centrifuged at 19987 RCF

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(×g) for 40 min at 4 ºC to obtain the RIF loaded polymer NPs. The supernatant solution was removed, and the NPs were washed with Millipore water three

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times. The RIF loaded polymer NPs were dried under vacuum overnight. The RIF loaded PLGA, PCP1, PCP2, PCP3, and PCP4 polymer NPs are referred to

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as PCPR0, PCPR1, PCPR2, PCPR3 and PCPR4 respectively (Table 1). (Yield: 300 mg - 420 mg; 60.0 - 84.0 %).

Preparation of INH loaded copolymer NPs

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INH loaded polymer NPs have been prepared by W/O/W double emulsification method as given in literature [10, 22]. Briefly, 400 mg of the copolymers (PCP series copolymers) were dissolved in 5 mL of DCM separately, and 3 mL of aqueous INH (150 mg) was added under ultra-

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sonication for 10 min using a probe-ultrsonicator (Hielscher- UP100H) to produce a W/O single emulsion. Then, 5 mL of aqueous PVA (0.5 % (w/v)) was

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added to the W/O single emulsion under magnetic stirring to get a W/O/W

double emulsion. After 30 min, 5 mL of DD water was added, and it was stirred

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magnetically for 2 h. The INH loaded polymer NPs dispersions were initially

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centrifuged at 4612 RCF (×g) to remove the bigger sized particles and then

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centrifuged at 19987.5 RCF (×g) for 40 min at 4 ºC to obtain INH loaded

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polymer NPs. Finally, the drug loaded polymer NPs were dried under vacuum

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overnight. The INH loaded PLGA, PCP1, PCP2, PCP3 and PCP4 polymer NPs will be referred to as PCPI0, PCPI1, PCPI2, PCPI3 and PCPI4 respectively.

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(Yield = 250 - 350 mg; 45 - 63.6 %).

Preparation of PYZ loaded copolymer NPs 400 mg) were dissolved in chloroform

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The PCP series copolymers (

and ultra-sonicated using a probe sonicator (Hielscher- UP100H) with 10 mL of aqueous PYZ solution (300 mg) for 15 min to get W/O primary emulsion [22]. Then, 5 mL of aqueous PVA (0.5 % (w/v)) was added to the primary emulsion and was stirred magnetically for 20 min for the formation of W/O/W double 9

emulsion. 5 mL of Millipore water was added to the W/O/W double emulsion and then it was stirred magnetically for 2 h. The PYZ loaded polymer NPs were initially centrifuged at 4612 RCF (×g) to remove the bigger sized particles and then centrifuged at 19987.5 RCF (×g) for 40 min at 4 ºC to obtain PYZ loaded

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polymer NPs. The INH loaded polymer NPs were dried under vacuum overnight. The PYZ loaded PLGA, PCP1, PCP2, PCP3 and PCP4 polymer NPs

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will be referred to as PCPZ0, PCPZ1, PCPZ2, PCPZ3 and PCPZ4 respectively.

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(Yield: 300 - 470 mg ; 42 - 57 %).

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Determination of RIF loading efficiency and percentage RIF content of RIF

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loaded polymer NPs

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RIF loaded polymer NPs (5 mg) were dissolved in 25 mL of chloroform

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and made up to 50 mL. The amount of RIF present in the solution was quantitatively determined by UV-Visible absorption spectrophotometry at 475

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calculated as:

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nm [23]. The drug loading efficiency and percentage drug content were

Amount of drug in NPs

x 100

Drug loading efficiency (%) (w/w) =

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Initial feeding amount of drug in formulation

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equation (1) Amount of drug in NPs Drug content (%) (w/w) = Total amount of NPs

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x 100

----------- equation (2)

Determination of INH loading efficiency and percentage INH content of INH loaded polymer NPs 5 mg of the INH loaded polymer NPs were ultra-sonicated for 20 min in

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10 mL of DD water and the solution was centrifuged at 19987 RCF (×g) for 30 min to remove the polymeric particles. The drug solution was transferred to a

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volumetric flask and the residue was washed with DD water. The centrifugate and washings were combined and made up to 50 mL. The amount of INH

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present in the solution was determined spectrophotometrically at 263 nm. The

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drug loading efficiency was calculated using the equations (1) and (2)

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respectively.

loaded polymer NPs

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Determination of PYZ loading efficiency and percentage PYZ content of PYZ

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5 mg of the PYZ loaded polymer NPs were ultra-sonicated by a probe

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sonicator (Hielscher- UP100H) for 20 min in 10 mL of phosphate buffered saline (PBS, pH 7.4). Then they were centrifuged at 19987 RCF (×g) for 30 min

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to remove the polymeric particles and washed thrice with the PBS. The drug solution was made up to 25-50 mL using the washings and PBS. The amount of PYZ present in the solution was determined spectrophotometrically at 269 nm. The drug loading efficiency and percentage drug content were calculated as shown in equations (1) and (2) respectively. 11

General method for the in vitro controlled release of tuberculosis drugs (RIF, INH, PYZ) 5 - 10 mg of RIF or INH or PYZ loaded PCP series polymeric NPs were

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dispersed in 5 mL of phosphate buffer saline (PBS) (pH=7.4) and incubated in rotary shaker at 37º [24]. Then they were centrifuged at 19987.5 RCF (×g) at

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predetermined time intervals. The amount of drug molecules released was

quantified by UV-Visible spectrophotometry. The drug loaded polymer NPs in centrifuge tube were again dispersed in 5 mL of freshly prepared PBS (pH=7.4)

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at each time interval to continue the drug release experiment. This procedure

Results and discussion

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Synthesis of copolymers

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was continued until the end of drug release from the polymer particles.

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The PLGA, CAP and Citrate-PEG-PLGA branched copolymers have been synthesized by direct melt condensation method (Fig. S1 and S2). The

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chemical structure of PLGA copolymer was confirmed by FTIR and 1H-NMR spectral analyses (Fig. 1 and Fig. 2). The physical properties of the PLGA such

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as melting point, inherent viscosity, number average and weight average molecular weight are tabulated (Table 1).

The ester bond formation and the chemical structure of the polymer have been confirmed by the FTIR and 1H-NMR spectral analyses. The FTIR 12

spectrum of CAP shows a strong absorption band at 1733 cm-1 which is not observed in the FTIR spectrum of PEG (Fig. 1a and b) indicating the formation of ester bond in the CAP copolymer [25]. The -CH stretching vibration of -CH2 groups of CAP molecule appears at a lower frequency region compared to that

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of PEG molecule. The 1H-NMR spectrum of CAP exhibits a signal at 2.8 ppm corresponding to -CH2 protons of citrate group, while it exhibits a signal at 3.6

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ppm corresponding to -CH2 protons of PEG molecule (Fig. 2A).[26] A signal at 4.2 ppm corresponds to the -CH2 protons of PEG which is connected to the

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citrate unit confirming the copolymerization of PEG and citric acid [21]. The

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methylene protons of PEG appear at 3.6 ppm while the methylene protons of

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CA units appear at 2.6 - 2.8 ppm. The chemical shift values of CA unit and PEG

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moiety of CAP in the present work are in accordance with that of reported

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values (Fig. 2A) [21].

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The FTIR spectrum of PLGA exhibits three absorption bands at 3004,

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2948 and 2893 cm-1 corresponding to the stretching frequencies of -CH3(LA), CH2(GA) and -CH(LA) respectively (Fig. 1c) [27]. When compared to the intensity

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of these bands with those of CA-PEG-PLGA multiblock copolymer (Fig. 1d), the intensity of -CH3 and -CH2 stretching frequencies are inverted. The intensity of CH2 stretching frequency is higher than that of -CH3 stretching frequency in the CA-PEG-PLGA multiblock copolymer due to the presence of excess -CH2 groups from PEG block, which is 13

reversed in the case of FTIR spectrum of PLGA. The FTIR spectrum of the PCP copolymer exhibits a peak at 1760 cm-1 corresponding to the ester carbonyl group [27]. The stretching frequency of ester carbonyl in the multiblock copolymer appears at higher frequency region (1760 cm-1) (Fig. 1d), when

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compared to that of CAP (1733 cm-1) (Fig. 1b). The two absorption bands observed at 1194 cm-1 and 1098

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cm-1 correspond to the -C-O stretching frequencies. The FTIR results confirm the presence of ester group in CA-PEG-PLGA multiblock copolymers.

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The 1H-NMR spectrum further confirms the chemical structure of CA-

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PEG-PLGA multiblock copolymer (Fig. 2C). The 1H-NMR spectrum of CA-

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PEG-PLGA multiblock copolymer exhibits complex signals at 4.2 - 4.7 ppm

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due to the -CH2 protons of GA. A signal at 5.2 ppm corresponds to -CH proton

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of LA, and a signal at 1.5 ppm corresponds to the -CH3 protons of LA. A signal at 2.8 ppm corresponds to the -CH2 protons of aconitate unit, while a signal at

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3.6 ppm corresponds to -CH2 protons of PEG moiety. Yao et al. have

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synthesized the oligomers of L-lactide-co-citric acid with ̅̅̅̅ Mn = 1346 - 2118 (g/mol) and found that the

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-CH2 protons of CA appear at 2.7 - 3.0 ppm and a quartet appear at 4.35 ppm due to -CH protons of lactate unit connecting citrate unit [28]. Similar results have been observed for the CA-PEG-PLGA multiblock copolymers in the present investigation (Fig. 2C). The 1

H-NMR spectral results indicate the presence of signals corresponding to both 14

CAP and PLGA copolymers which confirms the chemical structure of the CAPEG-PLGA multiblock copolymers. The PLGA and CAP copolymers melt at 102 and 42 oC respectively, while the PCP series branched copolymers melt in the temperature range of 45 -

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69 oC (Table 1). These results suggest that the melting point of PCP series branched copolymers decreases with increase in the concentration of PEG

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moiety (Table 1). The inherent viscosity of PLGA and CAP polymers is 0.14 and 0.17 g/dL respectively. The inherent viscosity of PCP series multi block

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copolymer varied from 0.18 g/dL to 0.24 g/dL.

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̅̅̅̅̅ of CAP was found to be 3573 (g/mol) indicating that during the The Mw

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melt polycondensation the macromonomer PEG6000 first underwent thermal

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depolymerization and then condensed with citric acid to produce the CAP. The

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thermal depolymerization of PEG is well-known in literature [29]. Hence, the molecular weight of CAP cannot be equal to the arithmetic sum of the

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̅̅̅̅̅ of PLGA was molecular weight of three PEG6000 and one CA units. The Mw

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2698 (g/mol) with polymer dispersity index (PDI) value 1.3 indicating a narrow ̅̅̅̅̅ of PCP series multi block molecular weight distribution of the PLGA. The Mw

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copolymers were in the range of 4443 - 5677 (g/mol). When compared to the PDI values of multiblock copolymers, the PCP1, PCP2 and PCP3 multiblock copolymers exhibited narrow molecular weight distribution (PDI = 1.3 - 1.35) while the PCP4 multiblock copolymer exhibited much wider molecular weight distribution (PDI = 6.04). These results indicate that the excess PEG could 15

induce depolymerization, if the amount of PEG used for copolymerization exceeds more than 1.2 g [30].

Microscopic analysis of drug loaded polymer NPs

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The FESEM images of the polymer NPs confirm the spherical shape of RIF loaded PCP series copolymer NPs. The RIF loaded polymer NPs exhibit a

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perfect spherical shape with a diameter of 220 - 270 nm (Fig. 3A). The INH (or) PYZ loaded polymer NPs exhibit spherical shape and the polymer particles

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joined together to form polymeric clusters (Fig. 3C and E). The W/O/W double

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emulsion technique could induce the formation of INH (or) PYZ loaded

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polymeric clusters. During W/O single emulsification the spherical particles

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form and in the second step (W/O/W emulsion) PVA interconnect the spherical

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polymer particles to produce drug loaded polymer clusters. In some cases, the spherical nature of the polymer particles get distorted due to melting of polymer

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by passage of electron beam during FESEM analysis.

TEM images of the RIF loaded PCP series copolymer NPs and PLGA

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MPs confirm the spherical shape of the particles (Fig. 4A and B). The TEM analysis indicates that the diameter of RIF loaded PCP series multiblock copolymer NPs is 200 - 450 nm, while the INH or PYZ loaded PCP series polymer NPs exhibit diameter in the range of 300 – 500 nm. These results

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suggest that W/O single emulsion technique produce individual NPs, while the W/O/W double emulsion method produce polymeric clusters.

Drug loading efficiency and percentage drug content of drug loaded polymer

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NPs The PCPR4 system with 49.2 % of PEG content exhibit 74.6 % of RIF

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loading efficiency and 20.2 % of RIF content, while the PCPR1 system with

19.5 % of PEG content in its copolymer backbone exhibit 50.8 % and 12.8 % of

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RIF loading efficiency and RIF content in the NPs respectively (Table 2). Diab

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et al. have reported the drug loading efficiency of PLGA MPs up to 34.2 %

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[31], while, Doan et al. have reported 5.6 % of RIF content in PLGA MPs [32].

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The effect of PEG on the preparation of RIF loaded PLGA MPs was

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investigated and it was found that the addition of PEG in the drug formulation increased the drug loading efficiency of RIF up to 66 % [33]. The earlier reports

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clearly indicated the importance of incorporation of PEG moiety into PLGA to

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increase the RIF loading efficiency. The PCPR series systems exhibited better RIF loading efficiency and RIF content due to their branched nature when

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compared to the literature values (Table 2) [34-37].

The UV-visible spectra of RIF exhibit absorption maximum at 475 nm, INH show absorption maximum at 263 nm, and PYZ show absorption maximum at 269 nm (Fig. 5) [38]. The PCP series NPs showed higher 17

absorbance than that of PLGA MPs in all the three drug formulations and the absorbance increases with increased PEG content in PCP series polymers (Fig. 5). The PLGA (PCPI0) MPs exhibit 10.4 % and 4.6 % of INH loading efficiency and percentage INH content, while these values increased up to 24.5

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and 13.6 % respectively for PCP series branched copolymer systems (Table 2). The PLGA (PCPZ0) MPs exhibit 10.2 and 3.3 % of PYZ loading efficiency and

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drug content respectively (Table 2). The PYZ loading efficiency of PCPZ series systems increased up to 25.4 - 57.3 %, while the PYZ content increased up to

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4.9 - 11.4 % by incorporating the PEG content in the branched PCP series

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copolymers. The earlier reports clearly indicated that it was difficult to load a

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hydrophilic drug on to a hydrophobic polymer matrix [39, 40]. Interestingly,

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branched PCP series polymer NPs exhibited higher drug loading efficiency and

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drug content when compared to those of PLGA-PEG-PLGA linear triblock

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copolymer NPs reported in the literature [10].

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In vitro drug release study

The RIF loaded PCPR series NPs showed 75 - 95 % of cumulative RIF

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release, while the RIF loaded PLGA MPs exhibited less than 20 % of cumulative RIF release up to 840 h (Fig. 6A). The histogram of in vitro release of RIF indicates that the PCPR series NPs exhibit an initial burst release followed by a sustained drug release (Fig. S3A). The PCPR2, PCPR3 and PCPR4 NPs exhibited in vitro release of RIF up to 264 h, while the PCPR1 NPs 18

showed the RIF release up to 840 h. The PCPR1 branched system exhibited sustained release of RIF for a prolonged period (840 h) when compared to that of PLGA-PEG based linear structures [11, 12, 16]. The in vitro cumulative INH release profiles of PCPI series polymer particles indicates that the PCPI0

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(PLGA) MPs released only 30 % of INH release up to 24 h (Fig. 6B). The PCPI1 polymer NPs exhibited 97 % of drug release up to 48 h. The PCPI2 NPs

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released 85.5 % of INH up to 48 h, while the PCPI3 NPs released 77 % of INH up to 96 h. These results indicate that the introduction of 47 % of PEG into the

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PCPI3 copolymer system increased the INH release time up to 96 h (Table 1)

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(Fig. 6). Since, the INH is highly water soluble, its rate of in vitro release in

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PBS is higher compared to that of RIF [10]. However, the rate of in vitro release

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of INH could be decreased by conjugating the polymer with gold nanoparticles

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[16].

The PYZ loaded PCPZ series polymer particles exhibited in vitro release

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of PYZ up to 720 h (Fig. 6C). The PLGA MPs exhibited 41 % of initial burst

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release within 5 h and the PYZ release from PCPZ0 MPs ceases within 48 h. The PCPZ1 NPs showed 79.1 % of initial burst release within 5 h and exhibited

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a sustained release up to 720 h, while the PCPZ2 NPs exhibited 41.8 % of initial burst release followed by sustained drug release up to 720 h. It is interesting to note that the PCPZ2 NPs exhibited better in vitro drug releasing property for a prolonged period (720 h) when compared to that of PLGA-PEG based linear multiblock copolymer systems (264 h) [16]. The histogram of amount of PYZ 19

released at different time durations indicates that the PCPZ2 NPs system showed maximum drug release at all time intervals after the initial burst release (Fig. S3C).

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The PLGA MPs showed relatively very little amount of RIF release and it was probably due to its lower rate of erosion. The PLGA usually exhibits RIF

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release by polymer erosion mechanism and the RIF entrapped inside PLGA

polymer matrix is released only after the erosion of PLGA [41]. Hence, the rate

U

of RIF release depends on the rate of erosion of PLGA. Since the rate of PLGA

N

erosion is a slower process, it releases only lesser amount of RIF. The rate of

A

polymer erosion can be modified by the introduction of hydrophilic moiety PEG

M

with PLGA [42].

ED

In the present study, the drug molecules are attached with the polymer matrix by hydrophobic or hydrophilic interactions. The hydrophobic PLGA

PT

showed lesser burst release of RIF. Since, the RIF is hydrophobic in nature, it is

CC E

strongly attached on surface of PLGA particles, and hence the PLGA MPs exhibit lesser burst release [11]. Since, the citrate-PEG-PLGA branched

A

amphiphilic copolymer NPs has lesser hydrophobicity than PLGA, the RIF binds weakly on surface of PCP series amphiphilic copolymer NPs resulting in higher burst release of RIF compared to PLGA MPs. Since, the INH and PYZ are hydrophilic in nature, only lesser amount of INH and PYZ could be loaded on surface of hydrophobic PLGA MPs resulting in lesser burst release [10]. 20

Alternatively, the citrate-PEG-PLGA amphiphilic copolymers could load higher amount of INH or PYZ due to an enhanced hydrophilicity on surface of citratePEG-PLGA amphiphilic copolymer NPs which could result in higher burst release of INH and PYZ in PBS compared to PLGA MPs. Even though the

IP T

PLGA MPs showed lesser burst release of TB drugs, they did not show sustained drug release after the initial burst release. The citrate-PEG-PLGA

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branched amphiphilic copolymers not only released higher burst release but also, they showed sustained drug release for prolonged period.

U

The drug release at the therapeutic level is very important because severe

A

N

side effects such as seizures, hepatotoxicity, deep metabolic acidosis and coma

M

by over dosage of TB drug have been reported [43]. Hence, the controlled release of TB drug within therapeutic level is necessary to avoid side effects.

ED

The branched type of PCP series copolymer NPs acted as potential controlled

PT

drug delivery systems for both hydrophobic and hydrophilic TB drug because of their amphiphilic nature. The PCP2 branched polymer system showed better in

CC E

vitro drug releasing property for INH and PYZ, while PCPZ1 system acted as a potential candidate for prolonged release of RIF compared to other polymer

A

systems.

Conclusions

21

A series of PLGA-PEG-citrate branched block copolymers was synthesized by melt condensation of PLGA (79:21) and CAP. The firs-line TB drugs RIF, INH or PYZ loaded branched PCP series copolymer NPs were prepared, and their drug release properties were investigated. The drug loaded

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amphiphilic PCP series polymer NPs showed higher drug loading efficiency and drug content percentage due to their branched structure when compared to those

SC R

of linear PLGA MPs and PLGA-PEG-PLGA triblock copolymer NPs. The

PCP2 branched copolymer system with a molar ratio of 54.7:13.6:31.7 (DL-

U

LA:GA:PEG) could be used as a potential drug carrier for the sustained release

N

of hydrophilic drugs INH and PYZ, while the PCP1 branched copolymer system

A

with a molar ratio of 62:18.5:19.5 (DL-LA:GA:PEG) could be used as a

M

potential candidate for the controlled release of RIF for a prolonged period

ED

within the therapeutic level. The present study provides a synthetic approach to design a branched type PLGA-PEG based amphiphilic copolymer system with a

CC E

drug.

PT

desired ratio of hydrophobic and hydrophilic moiety depending on the nature of

A

Disclosure

The authors disclose no conflicts of interest in this work

Acknowledgments

22

This research work was partly supported by Incheon National University through

A

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PT

ED

M

A

N

U

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Post-doctoral research program (2018-2019).

23

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PT

ED

M

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U

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List of Figure Captions Fig. 1. FTIR spectra of (a) PEG, (b) CAP, (c) PLGA and (d) PCP1 multiblock

M

A

N

U

SC R

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copolymer.

A

CC E

PT

ED

Fig. 2. 1H-NMR spectra of (A) CAP, (B) PLGA and (C) PCP2 copolymers.

28

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Fig. 3. FESEM images of (A) PCPR2, (B) PCPR0, (C) PCPI2, (D) PCPI0, (E) PCPZ2 and (F) PCPZ0 particles.

29

IP T SC R

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Fig. 4. TEM images of (A) PCPR2, (B) PCPR0, (C) PCPI2, (D) PCPI0, (E)

A

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PT

ED

M

A

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PCPZ2, and (F) PCPZ0 particles.

Fig. 5. UV-Visible absorption spectra of (A) RIF, (B) INH and (C) PYZ present in PCP series polymer NPs.

30

IP T SC R U N A

A

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PT

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PCP series polymer particles.

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Fig. 6. Cumulative drug release profiles of (A) RIF, (B) INH and (C) PYZ from

31

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A ED

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A

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Table 1. Physical properties of PCP series polymers PLGAa 1987

CAP PCP1c b 1588 3669

PCP2c 4102

PCP3c 4110

PCP4c 735

2698

3573 4928

5552

5677

4443

1.3 0.14

2.2 0.17

1.35 0.18

1.3 0.24

6.04 0.23

102

56-60

52-56

48-50

Yield

70 %

4064-66 43 80 % 3.3(g)

2.91(g)

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1.3 0.19

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Properti es ̅̅̅̅̅ 𝐌𝐧 (g/mol) ̅̅̅̅̅ 𝐌𝐰 (g/mol) PDI [η]* (dL/g) mp (°C)

2.87(g)

3.53(g)

A

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PT

ED

M

A

N

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62:18.5 54.7:13. 17.8:34. 15.6:34. Ratio of 79:21 :19.5 6:31.7 5:47.7 4:49.2 LA:GA: PEG *Inherent viscosity a PLGA(79:21) b Citrate-PEG copolymer c PCP1, PCP2, PCP3 and PCP4 are Citrate-PEG-PLGA branched copolymers synthesized with 0.4 g, 0.8 g, 1.2 g and 1.6 g of CAP respectively.

33

Table 2. Drug loading efficiency and percentage drug content of drug loaded PCP series polymer NPs. Drug content (%) RIF INH PYZ

Samples PLGA

29.2

10.4

10.2

6.1

4.6

3.3

PCP1

50.8

14.7

25.4

12.8

6.3

4.9

PCP2

51.3

16.7

32.1

13.4

7.1

8.1

PCP3

62.2

19.4

45.2

19.0

PCP4

74.6

24.5

57.3

20.2

N A M ED PT CC E A

34

SC R

IP T

Drug loading efficiency (%) RIF INH PYZ

8.3

9.8

13.6

11.4

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Polymer