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Sep 22, 2018 - (CH5-PAA) for the formulation of a novel bisnaphthalimide compound [16]. ... exerts its effects via inhibition of thymidylate synthase (TS) and the ...... Förster, A.; Markus, A. Amphiphilic Block Copolymers in Structure Controlled.
pharmaceuticals Article

A Novel PAA Derivative with Enhanced Drug Efficacy in Pancreatic Cancer Cell Lines Ali Alsuraifi 1,2 , Paul Kong Thoo Lin 3 , Anthony Curtis 1 , Dimitrios A. Lamprou 4, * Clare Hoskins 1, * 1 2 3 4

*

and

Institute of Science and Technology in Medicine, Keele University, Keele ST5 5BG, UK; [email protected] (A.A.); [email protected] (A.C.) College of Dentistry, University of Basrah, Basrah 61004, Iraq School of Pharmacy and Life Sciences, Robert Gordon University, Aberdeen AB10 7GJ, UK; [email protected] School of Pharmacy, Queen’s University Belfast, Belfast BT9 7BL, UK Correspondence: [email protected] (D.A.L.); [email protected] (C.H.); Tel.: +44-28-9097-2617 (D.A.L.); +44-17-8273-4799 (C.H.)

Received: 8 August 2018; Accepted: 19 September 2018; Published: 22 September 2018

 

Abstract: Nanoparticles have been shown to be effective drug carriers in cancer therapy. Pancreatic cancer forms dense tumours which are often resistant to drug molecules. In order to overcome such multidrug resistance, new drug entities, novel delivery systems and combination therapy strategies are being explored. In this paper, we report the design and synthesis of a poly(allylamine)-based amphiphile modified with hydrophobic naphthalimido pendant groups. Bisnaphthalimide compounds have been shown to possess anticancer activity. The potential of this polymer to encapsulate, solubilize and enhance drug (5-fluorouricil and bis-(naphthalimidopropyl)-diaminooctane) cytotoxicity in BxPC-3 cells was evaluated. Our studies showed that the insoluble drugs could be formulated up to 4.3 mg mL−1 and 2.4 mg mL−1 inside the amphiphiles, respectively. Additionally, the novel poly(allylamine)-naphthalimide carrier resulted in an amplification of cytotoxic effect with drug treatment after 24 h, and was capable of reduction of 50% cell population at concentrations as low as 3 µg mL−1 . Keywords: nanomedicine; nanopharmaceutics; drug solubilisation; pancreatic cancer; bisnaphthalimide

1. Introduction Poor aqueous solubility or dissolution of active ingredients is one of the most significant problems hindering effective drug delivery. More than 40% of new drugs under investigation have poor water solubility and hence poor bioavailability [1,2]. A number of solubility-enhancing strategies including co-solvents, micronization and nanonisation, amorphous solid dispersions (ASDs) [3], co-crystal formation [4], surfactants [5], complexation utilizing cyclodextrins [6] and the use of polymers [7–10] have been utilized to address this problem. Amphiphilic polymers have been developed as a first-rate alternative to low molecule weight surfactants for drug solubilisation. This is due to the lower excipient:drug ratios required for solubilisation and a higher degree of stability due to the decreased critical aggregation concentrations [11]. The most unique characteristic of amphiphilic polymers is their wide array of structures and architectures [11,12]. Amphiphilic polymers may exist as block copolymers [13], graft polymers [11], dendrimers [14] and star shaped polymers [15]. Each architecture has different physical properties, but all can be used to solubilise hydrophobic compounds. Amphiphilic poly(allylamine) (PAA) derivatives have been explored for their potential in drug delivery [16–19]. Hoskins and colleagues assessed the in vitro and in vivo pancreatic anticancer Pharmaceuticals 2018, 11, 91; doi:10.3390/ph11040091

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Amphiphilic poly(allylamine) (PAA) derivatives have been explored for their potential in drug delivery [16–19]. Hoskins and colleagues assessed the in vitro and in vivo pancreatic anticancer action of a nano-sized novel poly(allylamine) derivative graftedgrafted with 5% cholesteryl pendant groupsgroups (CH5action of a nano-sized novel poly(allylamine) derivative with 5% cholesteryl pendant PAA) for thefor formulation of a novel compound [16]. Bisnaphthalimide based drugs (CH5 -PAA) the formulation of abisnaphthalimide novel bisnaphthalimide compound [16]. Bisnaphthalimide based act as act DNA intercalators and have shown huge potential in in pancreatic cancer drugs as DNA intercalators and have shown huge potential pancreatic cancertherapy therapy[16,20]. [16,20]. However, theiractivity activityis is such increased potency, decreased drug solubility is However, their such thatthat withwith increased drug drug potency, decreased drug solubility is observed. observed. Hence careful formulation are required to these renderpotent these potent anticancer Hence careful formulation strategiesstrategies are required in orderin toorder render anticancer agents agents as clinically useable. Hoskins reported that after formulationinto intothe the CH CH55-PAA as clinically useable. Hoskins reported that after formulation -PAA amphiphiles, amphiphiles, bis(naphthalimidopropyl)diaminooctane in in solubility enhancement up to bis(naphthalimidopropyl)diaminooctane(BNIPDaoct) (BNIPDaoct)resulted resulted solubility enhancement up0.3 to − 1 −1 mg mLmL[16].[16]. TheThe CHCH 5-PAA diddid notnot demonstrate any significant 0.3 mg demonstrate any significanttoxicity, toxicity,yet yetthe the formulation formulation 5 -PAA demonstrated demonstrated strong strong in in vitro vitro and and in in vivo vivo anticancer anticancer action action [16]. [16]. Here and evaluation evaluation of of aanovel novelpoly(allylamine) poly(allylamine)(PAA) (PAA)derivative derivativewhich whichis Here we we report report the the synthesis synthesis and is grafted with 5% naphthalimide moieties The hydrophobic moieties used, are grafted with 5% naphthalimide moieties (Figure(Figure 1a). The1(a)). hydrophobic moieties used, are envisaged to envisaged to allow for greater insertion andthe stability within core the through hydrophobic core through π-π allow for greater insertion and stability within hydrophobic π-π interactions between interactions between the BNIPDaoct molecules 1(b)) and the planar naphthalimide the BNIPDaoct drug molecules (Figure drug 1b) and the planar(Figure naphthalimide pendant groups. Additionally, pendant Additionally,pendant the usegroups of thewill naphthalimide will activity confer to some the use ofgroups. the naphthalimide confer some pendant inherent groups anti-cancer the inherent anti-cancer thedelivery polymer itself. biocompatibility Ideally, for most drugHowever, deliveryinvehicles, polymer itself. Ideally,activity for mostto drug vehicles, is vital. diseases biocompatibility is vital. However, in treatment diseases such pancreatictherapies cancer, combined treatment and such as pancreatic cancer, combined andas innovative are required in order to innovative therapies are required indrug orderpenetration. to overcome drug resistance and drug overcome drug resistance and poor Nanotechnologies havepoor shown to penetration. be useful for Nanotechnologies have shown be useful for this. As in such, propose that a synergistic reduction this. As such, we propose that to a synergistic reduction cellwe viability will occur after formulation in in cell viability will occur after in this system. It is proposed that these drug carriers if this system. It is proposed thatformulation these drug carriers if successful can be further functionalised with successful can be further functionalised with targeting moieties in order to bring the payload cargo targeting moieties in order to bring the payload cargo to the exact site hence reducing likelihood of to the exact site hence reducing likelihood of systemic toxicity. systemic toxicity. (a)

O O

O H2N

OH

N

Refluxing EtOH

O

(1)

TsCl, Pyridine

OH

O

O N O

OTs

Poly(allylamine), Cs2CO3

] 5%

[

NH H2N

(2)

H2N

H2N

H2N

(3) O

(b)

N

O

O N O

O

H N

N H

2HBr

N O

Figure 1. 1. Chemical of of poly(allylamine)-propylnaphthalimide (PAA-N) and Figure Chemical structures structuresfor for(a) (a)synthesis synthesis poly(allylamine)-propylnaphthalimide (PAA-N) (b) bis(naphthalimidopropyl)diaminooctane (BNIPDaoct). and (b) bis(naphthalimidopropyl)diaminooctane (BNIPDaoct).

The aggregation ability in aqueous environments and the ability to act as a drug solubilising agent The aggregation ability in aqueous environments and the ability to act as a drug solubilising was determined using novel BNIPDaoct and 5-fluorouricil (5-FU). 5-FU is an anti-cancer drug, which agent was determined using novel BNIPDaoct and 5-fluorouricil (5-FU). 5-FU is an anti-cancer drug, exerts its effects via inhibition of thymidylate synthase (TS) and the incorporation of its metabolites which exerts its effects via inhibition of thymidylate synthase (TS) and the incorporation of its into RNA and DNA [21]. Numerous studies have demonstrated the potential use of 5-FU in pancreatic metabolites into RNA and DNA [21]. Numerous studies have demonstrated the potential use of 5cancer as single therapy or in combination with the former gold standard treatment gemcitabine [22]. FU in pancreatic cancer as single therapy or in combination with the former gold standard treatment This study will evaluate whether the novel formulations formed, are capable of enhanced drug efficacy gemcitabine [22]. This study will evaluate whether the novel formulations formed, are capable of in vitro compared with the free drugs. enhanced drug efficacy in vitro compared with the free drugs.

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Results 2.2.Results 2.1.Synthesis Synthesisand andCharacterisation CharacterisationofofPolymer Polymer 2.1. Thestructures structures of of intermediates intermediates and and products products were were analysed analysed by by 11H-NMR H-NMR (Figure (Figure 2). 2). Peak Peak The 1 1 assignments for the H-NMR spectra of the naphthalimido alcohol product 1 were as follows: 2 ppm assignments for the H-NMR spectra of the naphthalimido alcohol product 1 were as follows: 2 ppm alkyl chain CH , 3.6 ppm alkyl chain CH beside an O atom and O-H, 4.4 ppm alkyl chain CH2 2 ppm alkyl chain CH2 beside 2 alkyl chain CH2, 3.6 an O atom and O-H, 4.4 ppm alkyl chain CH2 beside N atom and 7.8–8.6 ppm protons aromatic protons (Figure 2a). The O-tosyl (Figure spectrum (Figure 2b) abeside N atoma and 7.8–8.6 ppm aromatic (Figure 2(a)). The O-tosyl spectrum 2(b)) showed showed assigned peaks at 2.2 ppm alkyl chain CH , 2.5 ppm CH attached to an aromatic ring, 2 3 attached to an 3 aromatic ring, 4.3 ppm alkyl assigned peaks at 2.2 ppm alkyl chain CH2, 2.5 ppm CH 4.3 ppm chain CH2N s beside and N atoms 7.3–8.5protons. ppm aromatic protons. Thecharacteristic PAA showed chain CH2alkyl s beside O and atoms O and 7.3–8.5 ppmand aromatic The PAA showed characteristic peaks at2,1.3 ppm CHto -NH(Figure (Figure 2c). 2 , 1.7 2 attached 2 of PAA2(c)). peaks at 1.3 ppm CH 1.7ppm ppmCH CH, 2.2ppm ppmCH, CH2.2 2 attached -NH2 oftoPAA After After modification with the naphthalimide group (PAA-propylnaphthalimide, Figure 2d) the spectrum modification with the naphthalimide group (PAA-propylnaphthalimide, Figure 2(d)) the spectrum showedadditional additionalaromatic aromaticproton protonpeaks peaksatat7.3–8.5 7.3–8.5ppm, ppm,while whilethe theremaining remainingpropylnaphthalimide propylnaphthalimide showed peaks were overlapped with the PAA peaks. The peak at 3.3 ppm was due to water. peaks were overlapped with the PAA peaks. The peak at 3.3 ppm was due to water. (a)

(b)

(c)

(d)

1 H-NMR spectra of (a) compound 1 (Figure 1) in CDCl (b) compound 2 (Figure 1) in CDCl , Figure2.2.1H-NMR Figure spectra of (a) compound 1 (Figure 1) in CDCl3 3(b) compound 2 (Figure 1) in CDCl3,3 ◦ C. (c)PAA PAAin inDD2O and(d) (d)PAA-N PAA-Nin inDD2O carriedout outusing using300 300MHz MHzNMR NMRatat25 25°C. (c) 2 Oand 2 Ocarried

−1 that TheFTIR FTIRspectrum spectrum compound 1, (Figure S1a) displays a strong band at 3424 The of of compound 1, (Figure S1(a)) displays a strong band at 3424 cm−1cm that was was assigned thestretch, O-H stretch, in addition to a strong carbonyl absorption 1640 cm−1 −1 which assigned to theto O-H in addition to a strong carbonyl group group absorption at 1640atcm which confirms the synthesis was successful. the spectra for compound the disappearance confirms the synthesis was successful. In the In spectra for compound 2 the 2disappearance of theof the hydroxyl group band suggests the esterification between compound 1 and para-toluenesulfonyl hydroxyl group band suggests the esterification between compound 1 and para-toluenesulfonyl chlorideoccurred occurred(Figure (Figure S1(b)). S1b). The spectra (Figure S1c) showed characteristic peaks at 3377 cm−1 chloride ThePAA PAA spectra (Figure S1(c)) showed characteristic peaks at 3377 and 2846 2846 cm−1cm due to the the in polymer backbone. PAA-NPAA-N (Figure (Figure S1d) possessed −1 and −1 due cm toN-H the and N-HC-H andinC-H the polymer backbone. S1(d)) −1 and similar peaks to the PAA alone. However, the disappearance of strong peaks between 1410 cm possessed similar peaks to the PAA alone. However, the disappearance of strong peaks between 1410 1 , which related to the sulfonyl of the para-toluenesulfonyl group, confirms that the coupling 1177 cm−1177 −1 and cm cm−1, which related to the sulfonyl of the para-toluenesulfonyl group, confirms that the had been successful. coupling had been successful.

2.2. Characterisation of Nano-Aggregates 2.2. Characterisation of Nano-Aggregates Hydrodynamic diameter, polydispersity index (PDI) and zeta (ζ) potential were measured for the Hydrodynamic diameter, polydispersity index (PDI) and zeta (ζ) potential were measured for PAA-N aggregates in aqueous solution. The data showed that the self-assemblies formed possessed a the PAA-N aggregates in aqueous solution. The data showed that the self-assemblies formed possessed a hydrodynamic diameter of 366  14 nm at 1 mg mL−1 (Figure 3(a)). The polydispersity of

the aggregates formed was 0.37 (Figure 3(b)), this indicated that the aggregates formed were relatively mono-disperse in solution. The particle size (after storage in solution) was monitored over a 4-week period (Table S1), no significant change in size was determined rendering the particles stable over this timeframe. The amphiphiles possessed a positive surface charge in aqueous solution Pharmaceuticals 2018, 11, 91 4 of 13 of +73  1 mV (Figure 3(c)). This charge is attributed to the high primary amine content in the backbone of the PAA. −1 order Surface tension used the critical hydrodynamic diametermeasurement of 366 ± 14 nmwas at 1 mg mLin (Figureto 3a).determine The polydispersity of theaggregation aggregates concentration (CAC) in aqueous media of the The surface tension graph for formed was 0.37 (Figure 3b), this indicated thatself-assemblies the aggregates formed. formed were relatively mono-disperse PAA-N (Figure 3(d)) showed that the surface tension remains unchanged from that of water (0.00726 in solution. The particle size (after storage in solution) was monitored over a 4-week period (Table S1), −1) at very low concentrations (up to 0.065 mg mL−1). However, with increasing polymer Nm no significant change in size was determined rendering the particles stable over this timeframe. The concentration, surface tension decreased dramatically. Thesolution CAC value was the amphiphiles possessed a positive surface charge in aqueous of +73 ± 1determined mV (Figure from 3c). This −1. surface tension graph to be 0.125 mg mL charge is attributed to the high primary amine content in the backbone of the PAA.

(a)

(b)

PAA-N-BNIPDaoct

PAA-N-BNIPDaoct

PAA-N-5FU

PAA-N-5FU

PAA-N

PAA-N 0

(c)

100

200 300 Size, nm

400

0

0.2

0.4

0.6

PDI

(d) Surface Tension Nm-1

0.075 PAA-N-BNIPDaoct PAA-N-5FU PAA-N -20

30 Zeta potential, mV

80

0.07 0.065 0.06 0.055 0.001 0.01 0.1 1 Polymer concentration, mgmL-1

10

Figure 3. 3. Characterisation (a) photon photon correlation correlation Figure Characterisation of of the the nano-aggregates nano-aggregates in in aqueous aqueous media media via via (a) spectroscopy and and (b) (b) polydispersity polydispersityindex, index,(c) (c)zeta zetapotential potentialmeasurement measurement(n(n==3,3,±  SD) spectroscopy SD) and and (d) (d) surface surface tension measurement. measurement. tension

Surface tensionof measurement was used in order to determine the criticalhad aggregation concentration TEM imaging the PAA-N (Figure S2(a)) showed nano-aggregates been formed. The size (CAC) aqueous was media of the self-assemblies formed. Thecorrelation surface tension graph for of thesein aggregates notably smaller compared to the photon spectroscopy. ThisPAA-N is due −1 (Figure 3d) showed that the surface tension remains unchanged from that of water (0.00726 Nm to the discrete differences in technique and measurement: photon correlation spectroscopy measures) −1 at low concentrations (up in to 0.065 mg mL withinteraction increasing of polymer concentration, thevery hydrodynamic diameter solution, which). However, involves the the polymeric selfsurface tension dramatically. The CAC from theofsurface tension assemblies with decreased the water molecules, whereas TEM value allowswas for determined direct measurement the aggregates graph to be 0.125 mg mL−1 . in a solid state. TEM imaging of the PAA-N (Figure S2a) showed nano-aggregates had been formed. The size of these aggregates 2.3. Drug Loadingwas and notably Release smaller compared to the photon correlation spectroscopy. This is due to the discrete differences in technique and measurement: photon correlation spectroscopy measures the Drugs (5-FU and BNIPDaoct) were loaded into the core of the self-assemblies via probe hydrodynamic diameter in solution, which involves the interaction of the polymeric self-assemblies sonication. The amount of drug encapsulated was quantified using UV-Vis spectroscopy and HPLC. with the water molecules, whereas TEM allows for direct measurement of the aggregates in−1 a solid state. Figure 4 shows that the PAA-N self-assemblies were capable of solubilizing 2.4 mg mL and 4.3 mg mL−1Drug of BNIPDacot and 5-FU, respectively (Figure 4(a)). The difference in drug incorporation ability 2.3. Loading and Release between the two drugs is possibly due to difference in molecular weight. The 5-FU exhibited an Drugs (5-FU and BNIPDaoct) werethe loaded into thewas coreencapsulated of the self-assemblies viathe probe sonication. encapsulation efficiency of 17% whilst BNIPDaoct at 10% into PAA-N nanoThe amount of drug encapsulated was quantified using UV-Vis spectroscopy and HPLC. aggregates. The 5-FU is a smaller molecule which may have resulted in greater quantities physically Figure 4 shows thatspace the PAA-N were solubilizing 2.4 mg after mL−1drug and fitting within the core of the self-assemblies nano-aggregates. Thecapable size ofofthe nano-aggregates − 1 4.3 mg mL of BNIPDacot and 5-FU, respectively (Figure 4a). The difference in drug incorporation loading was determined via photon correlation spectroscopy (Figure 3(a)) and TEM imaging (Figures ability between the two drugs is possibly due to difference in molecular weight. The 5-FU exhibited an encapsulation efficiency of 17% whilst the BNIPDaoct was encapsulated at 10% into the PAA-N nano-aggregates. The 5-FU is a smaller molecule which may have resulted in greater quantities physically fitting within the core space of the nano-aggregates. The size of the nano-aggregates after drug loading was determined via photon correlation spectroscopy (Figure 3a) and TEM imaging

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(Figure Here the loaded particles appeared to undergo core compaction after drug with loading S2(a,b)).S2a,b). Here the loaded particles appeared to undergo core compaction after drug loading the with the hydrodynamic radius reducing nm PAA-N of the PAA-N nm220 andnm 220after nm after hydrodynamic radius reducing from 366from nm 366 of the to 159 to nm159 and drug drug incorporation and BNIPDaoct respectively) (Figure This is likely increase incorporation (5-FU(5-FU and BNIPDaoct respectively) (Figure 3(a)).3a). This is likely duedue to to thethe increase in in hydrophobicity after drugincorporation incorporationpulling pullingthe thependant pendantgroups groupscloser, closer, resulting resulting in in aa tightly hydrophobicity after drug tightly packed packed compact compactaggregate. aggregate. This This trend trend was was consistent consistent with with the the smaller smaller aggregates aggregates also also being being observed observed in Stability studies over a 4-week period indicated no no change in in the the TEM TEM micrographs micrographs (Figure (FiguresS2b,c). S2(b,c)). Stability studies over a 4-week period indicated change particle size, as measured by photon correlation spectroscopy (Table S1).S1). in particle size, as measured by photon correlation spectroscopy (Table

(a)

(b)

*

2 1 0 PAA-N-BNIPDacot PAA-N-5FU Formulation

% Drug release

Amount of drug solubilised, mgmL-1

4 3

100

*

5

PAA-N-BNIPDaoct

80

PAA-N-5FU

60 40 20 0 0

200

400

600 800 1000 1200 1400 Time, mins

Figure Drugloading loading(a) (a)and andrelease release studies 5-FU BNIPDaoct PAA-N Figure 4. 4. Drug (b)(b) studies of of 5-FU andand BNIPDaoct intointo and and fromfrom PAA-N selfself-assemblies (n = 3, ± SD). * denotes a significant increase in aqueous solubility compared assemblies (n = 3,  SD). * denotes a significant increase in aqueous solubility compared to to the the unformulated drug (p < 0.01). Studies were quantified using UV-Vis spectroscopy (5-FU) and HPLC unformulated drug (p < 0.01). Studies were quantified using UV-Vis spectroscopy (5-FU) and HPLC (BNIPDoact). (BNIPDoact).

It is not expected that drug loading into the core of polymeric self-assemblies should result It is not expected that drug loading into the core of polymeric self-assemblies should result in a in a change in zeta potential measurement. This is due to the fact that the surface charge of the change in zeta potential measurement. This is due to the fact that the surface charge of the aggregates aggregates should remain unchanged if a drug has been successfully incorporated into the core. As should remain unchanged if a drug has been successfully incorporated into the core. As evident in evident in Figure 3c, incorporation of 5-FU into the PAA-N did not result in any significant changes in Figure 3(c), incorporation of 5-FU into the PAA-N did not result in any significant changes in surface surface charge. However, the data from the BNIPDaoct loaded PAA-N aggregates showed a dramatic charge. However, the data from the BNIPDaoct loaded PAA-N aggregates showed a dramatic reduction in surface charge from +73 mV to +3.6 mV. The reason for this is not known. The BNIPDaoct reduction in surface charge from +73 mV to +3.6 mV. The reason for this is not known. The BNIPDaoct is a polyamine-based drug and hence when protonated should have an overall net positive charge is a polyamine-based drug and hence when protonated should have an overall net positive charge and, therefore, should not form charge-charge complexes with the cationic PAA-N. However, the and, therefore, should not form charge-charge complexes with the cationic PAA-N. However, the naphthalimide moieties at each end of the polyamine chain do carry residues with a high electron naphthalimide moieties at each end of the polyamine chain do carry residues with a high electron density. Therefore it may be possible that the drug molecules BNIPDaoct, have become anchored density. Therefore it may be possible that the drug molecules BNIPDaoct, have become anchored into into the self-assemblies with one of the naphthalimido groups of the drug sitting exterior to the core. the self-assemblies with one of the naphthalimido groups of the drug sitting exterior to the core. Further investigation would be needed to confirm this theory. Further investigation would be needed to confirm this theory. Drug release studies (Figure 4b) were carried out in water at room temperature over 24 h. The data Drug release studies (Figure 4(b)) were carried out in water at room temperature over 24 h. The showed that the BNIPDaoct loaded aggregates discharged their drug payload more rapidly than the data showed that the BNIPDaoct loaded aggregates discharged their drug payload more rapidly than 5-FU loaded aggregates. Both formulations experienced 25% drug release within the first hour. After the 5-FU loaded aggregates. Both formulations experienced 25% drug release within the first hour. 24 h the BNIPDaoct had released 73% of drug compared with only 43% of the 5-FU being released. After 24 h the BNIPDaoct had released 73% of drug compared with only 43% of the 5-FU being This may have been due to the internalization mechanism of the drugs. Theoretically, the BNIPDaoct released. This may have been due to the internalization mechanism of the drugs. Theoretically, the would have been expected to have been retained within the hydrophobic core of the self-assemblies BNIPDaoct would have been expected to have been retained within the hydrophobic core of the selffor much longer than the 5-FU. This is due to the greater degree of hydrophobicity of the BNIPDaoct assemblies for much longer than the 5-FU. This is due to the greater degree of hydrophobicity of the compared with the 5-FU as evidenced by their respective intrinsic solubilities. Additionally, it was BNIPDaoct compared with the 5-FU as evidenced by their respective intrinsic solubilities. postulated that upon inserting within the hydrophobic core, the BNIPDaoct would be able to stack Additionally, it was postulated that upon inserting within the hydrophobic core, the BNIPDaoct in close proximity with little steric hindrance due to the planar nature of the molecule. However, would be able to stack in close proximity with little steric hindrance due to the planar nature of the given the large decrease in zeta potential observed (Figure 2a) after BNIPDaoct incorporation, this molecule. However, given the large decrease in zeta potential observed (Figure 2(a)) after BNIPDaoct may not be the case, and hence, the more rapid drug release compared to the smaller 5-FU molecule. incorporation, this may not be the case, and hence, the more rapid drug release compared to the This finding does support the suggestion that polymer-drug complexation is occurring which is more smaller 5-FU molecule. This finding does support the suggestion that polymer-drug complexation is rapidly broken down compared to the time taken for 5-FU to become un-encapsulated. occurring which is more rapidly broken down compared to the time taken for 5-FU to become unencapsulated.

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% Cell viability

2.4. Cytotoxic Activity 2.4. Cytotoxic Activity The cytotoxic effect of PAA-N, BNIPDaoct, 5-fluorouracil, PAA-N-BNIPDaoct and PAA-N-5FU The cytotoxic PAA-N, BNIPDaoct, 5-fluorouracil, PAA-N-BNIPDaoct andshowed PAA-N-5FU was determined by effect MTT of assay in BxPC-3 cells after 24 h exposure (Figure 5). The data that was determined by MTT assayin in any BxPC-3 cells after 24 h exposure (Figure 5). The data showed that both free drugs did not result significant decrease in cell viability over the concentrations both free drugs did not result in any significant decrease in cell viability over the concentrations tested. tested. 100 90 80 70 60 50 40 30 20 10 0

* * *

PAA-N 5-FU PAA-N-5FU BNIPDacot PAA-N-BNIPDaoct

* * *

*

0

*

*

0.02

* * *

* *

0.04

0.06

0.08

0.1

Drug concentration, mgmL-1 Figure 5. Cytotoxicity of 5-FU and BNIPDaoct loaded PAA-N in BxPC-3 cells after 24 h exposure (n = 3, Figure 5. Cytotoxicity of 5-FU and BNIPDaoct loaded PAA-N in BxPC-3 cells after 24 h exposure (n = ± SD). * denotes a significant reduction in viability compared with the control cells (p < 0.01). 3,  SD). * denotes a significant reduction in viability compared with the control cells (p < 0.01).

The PAA-N carrier possessed an IC50 value of approximately 28 µg mL−1 which was reduced −1 which was reduced to The carrier possessed an IC50 value of approximately 28 μg mL to 7.5 µgPAA-N mL−1 after 5-FU incorporation. The BNIPDaoct formulation was more potent with IC50 −1 after 5-FU incorporation. The BNIPDaoct formulation was more potent with IC50 7.5 μg mL − 1 − 1 observed at 3 µg mL (10-fold). Interestingly, 0.05 mg mL the PAA-N-5FU appeared less toxic than −1 (10-fold). Interestingly, 0.05 mgmL−1 the PAA-N-5FU appeared less toxic than observed 3 μg mL the emptyatcarrier (PAA-N). However, upon further increase in concentration this appeared similar. the empty carrier (PAA-N). However, upon further increase in concentration this appeared similar. 2.5. Cellular Uptake of Drug 2.5. Cellular Uptake of Drug Drug uptake in BxPC-3 cells was measured quantitatively using UV-Vis spectroscopy (5-FU) and Drug uptake in allowing BxPC-3 cells wasamount measured quantitatively UV-Vis spectroscopy (5-FU) HPLC (BNIPDoact) for the of drug per cell tousing be estimated. Figure 6a shows the and HPLC allowing amount per cell be5-FU estimated. Figure 6(a) shows amount of(BNIPDoact) 5-FU internalised intofor thethe cells, here itofisdrug evident thattothe encapsulated within the the amount of 5-FU internalised into the cells, here it is evident that the 5-FU encapsulated within the PAA-N resulted in significantly greater (p < 0.001) uptake compared to the free drug. The BNIPDaoct PAA-N in significantly greater (p