Synthesis and Degradation Study of Cationic Polycaprolactone-Based

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Synthesis and Degradation Study of Cationic PolyCaprolactonebased Nanoparticles for Biomedical and Industrial Applications Azzurra Agostini, Simone Gatti, Alberto Cesana, and Davide Moscatelli Ind. Eng. Chem. Res., Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 8, 2017

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Industrial & Engineering Chemistry Research

Synthesis and Degradation Study of Cationic PolyCaprolactone-based Nanoparticles for Biomedical and Industrial Applications Azzurra Agostini, Simone Gatti, Alberto Cesana and Davide Moscatelli *

Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Via Mancinelli 7 - 20131 Milano, Italy. *: Corresponding author: Davide Moscatelli; E-mail: [email protected], Phone: +39 02 2399 3135 ABSTRACT Positively charged polymers have increased their importance in the last years for the possibility to be used in many different applications, from gene delivery to polymer flooding applications and as flocculants in waste water treatment. In all cases, the possibility in obtaining biodegradable colloidal products leads to great advantages. In this work, positively charged NPs have been produced via free radical emulsion polymerization (FREP). This synthetic route was selected since it is widely used in industry and it facilitates the large scale production along with the control of some key features of the final NPs such as their size, surface charge and particle size distribution dispersity. NP synthesis was carried out by a four-step process: the synthesis of biodegradable esterbased macromonomers obtained through the ring opening polymerization (ROP) of the εcaprolactone (CL), the reaction among the obtained macromonomer and succinic anhydride, the final condensation with choline chloride to obtain the positively charged macromonomer and the FREP polymerization of the produced macromonomer. The effects of reaction conditions on NP characteristics were studied and the tunable behaviour of the obtained charged NPs has been proved, also in term of degradation time.

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KEYWORDS Degradable charged NPs, Polycations, Emulsion Polymerization, Hydrolytic Degradation 1. Introduction Biodegradable polycationic materials have already reached a significant importance in different and smart applications worldwide. Several industrial and clinical fields are involved in using cationic polymer due to their ability to form ionic bonds as to remove anionic compounds and to delivery ionic drugs inside the human body.1, 2 Size and particle size distribution (PSD) of particles made of cationic polymer are only two of the main requirements for biomedical applications. In order to obtain particles of a given size, with a narrow PSD, the choice of the polymerization process is a key step.3 The most suitable ways to synthesize polymeric colloidal particles are those related to heterophase polymerization processes, in particular the free radical emulsion polymerization (FREP).4 In fact, the FREP is the most efficient and profitable polymerization technique to produce polymeric NPs. The polymerization reaction is carried out in a continuous phase where monomer is slightly soluble in, reaction times are usually short and high conversions of monomer are obtained. The possibility of controlling both particle size distributions (and therefore their monodispersity) and, if necessary, molecular weight distributions, polymer structure, and surface functionalization is easily and promptly achieved. Furthermore, FREP is a widely used industrial process capable to produce, in safe conditions, huge amounts of dispersed polymers every year with several applications including adhesives, coatings, paper additives and textile modifiers.5 Among all the monomers which can be polymerized in this way, positively charged ones allows the synthesis of polycations that found applications in oral delivery, gene therapy, flocculation processes and polymer flooding. Oral delivery 2 ACS Paragon Plus Environment

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Different works show that polycations are potential tools for increasing drug solubility, protect labile compounds from pH changes and digestive enzymes, and serve as permeation enhancers for oral delivery applications.6,

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Furthermore, polycations are so interesting because of their

mucoadhesive properties, that improve the retention time of drugs in the gastro intestinal tract, and their ability to promote the absorption process by a variety of mechanisms.8 Gene therapy Nowadays, cationic based-polymer or liposomes are the most used non-viral vectors for delivery of different types of nucleotides for gene field due to the ability to uptake a significant amount of genetic material inside cell.9-13 Exogenous DNA/RNA must be protected from damage such as their fast enzymatic degradation in physiological condition and their entry into the cell has to be facilitated.14 In fact, polycations showed the ability to protect the drug from undesirable degradation during the transfection process. Because of their positive charges, cationic vectors allow an easy cellular uptake and the use of cationic micelles/nanoparticles have been commonly selected in gene therapy applications. Cationic nanocarriers enable endocytosis and ensure the entrapment of entities loads impermeable to the cell membrane, such as genetic molecules, and the successive release from the endosomes to the nucleus, where the genetic material can perform.15, 16 Flocculation A different field of application for polycations is flocculation processes.17 Wastewater treatment, mineral processing and paper making are some examples of the applications requiring a flocculation procedure.18-21 Usually, flocculants are obtained via copolymerization of acrylamide and cationic acrylates/methacrylates so as to separate negatively charged components.22, 23 The destabilization of the suspension occurs due to two mechanisms: first the neutralization of the charges and then, the particle bridging. In order to accelerate this process and to enhance its efficiency, cationic basedpolymers should have a high molecular weight and a high specific amount of superficial charges or have to be organized into positively charged colloids.24,

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In fact, the high molecular weight

facilitates particle bridging flocculation while the cationic charge provides neutralization. 3 ACS Paragon Plus Environment


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Polymer/ Surfactant Flooding Polycations play an important role in polymer flooding projects.26-30 The selected polymer must satisfy the following requirements: i) solubility in water, ii) good injectivity, iii) improving aqueous viscosity and iv) the solution must maintain a relatively high viscosity under the influences of water salinity and temperature. The presence of charges improves the water solubility of polymer allowing the use of positive charged colloids or cationic polymers with high molecular weight, both necessary to enhance the viscosity of the solution. In addition, amphiphilic cationic based-polymers has to be taken into account due to their ability to behave as tensides, suitable to recover the amount of oil adherent to the rock formation and then increasing the oil productivity in the so called EOR applications. 26, 31-33

In this work, FREP has been selected to obtain biodegradable and biocompatible cationic-based nanoparticles potentially used in many different applications. The biodegradable behaviour allows to realize eco-friendly systems for flocculation or polymer flooding processes, while biocompatibility is necessary for the uses in biomedical applications. Caprolactone-based methacrylates (reported as PCLnMA) are obtained via ROP due to the reaction of the monomer caprolactone (CL) with the co-catalyst 2-hydroxyethyl methacrylate (HEMA). The subsequent positively charged macromonomers (PCLnChMA) are synthesized through a three steps process reported in Scheme 1.


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Scheme 1: Synthesis to obtain biodegradable positively charged macromonomers (PCLnChMA) PCLnChMA plays the role of both cationic monomer and surfactant (i.e. it is a surfmer, a molecule which is able to stabilize the NPs and, at the same time, can be covalently incorporated into the polymer chains during polymerization due to the presence of a vinyl double bond).34,

35

Two

different FREP techniques are used to produce NPs: batch emulsion polymerization (BEP), in which a mixture of PCLnMA and PCLnChMA is put into the reactor at the beginning of the process, and monomer-starved semi-batch polymerization (MSSEP), in which PCLnChMA (hydrophilic monomer) is loaded into the reactor at the beginning while PCLnMA (lipophilic monomer) is fed slowly during the reaction in order to maintain the system in starved conditions with respect to the lipophilic monomer.36 The surfmer adopted, with a quaternary nitrogen, brings an important advantage in the final NPs, being stable in a large pH range and in different environmental 5 ACS Paragon Plus Environment


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conditions.37 The synthesis via FREP ensures to fine tune the NP size, the charge density and the ζ−potential. Finally, these NPs differ from other polymer-based materials since it is possible to control their degradation rate by changing the length of the CL side chains or, more in general, the polyester adopted, such as polylactic acid (PLA) and polylactic-co-glycolic acid (PLGA).38-42 2. Materials Alpha,alpha'-azodiisobutyramidindihydrochloride purum (αα-(+), Fluka, 98% purity), Aldrich), poly(ethylene glycol) methacrylate (PEGMA, Mn 2000 Da, 50%, Sigma Aldrich), ε-caprolactone (CL, 99%, Sigma Aldrich), 2-hydroxyethyl methacrylate (HEMA, containing up to 50 ppm of hydroquinone-monomethyl-ether, ≥99.9% purity, Sigma Aldrich), 2-ethylhexanoic acid tin(II) salt (Sn(Oct)2, ~95%, Sigma Aldrich), 4-(dimethylamino) pyridine (DMAP, ≥99% purity, Sigma Aldrich), N,N’-dycycloexylcarbodiimide (DCC, 99%, Sigma Aldrich), succinic anhydride (≥99% purity, Sigma Aldrich), choline chloride (≥99% purity, Sigma Aldrich), acetonitrile (ACN, 99.8%, Sigma Aldrich), deuterated chloroform (CDCl3, 99.8%, Sigma Aldrich), dimethyl sulfoxide-d6 (DMSO-d6, 99.9%, Sigma Aldrich) were all used as received. 2.1 PCLnMA Macromonomer Synthesis PCLnMA macromonomers (with n equal to 2, 3 and 5) were synthesized through ROP following a procedure reported elsewhere.

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CL was first heated up in a stirred flask. Temperature was

controlled by an external oil bath set to 130 ± 1 °C. A mixture of HEMA (1/n eq.) and Sn(Oct)2 (1/200n eq.) was added to the monomer to initiate the reaction which was carried out for 2.5 hours. At the end, macromonomers, without any further purification, were stored at 4°C. The obtained macromonomers were characterized via 1H-NMR, using a 500 MHz ultrashield NMR spectrometer (Bruker, Switzerland). For example, in the case of PCL5MA, 1H NMR (400 MHz, CDCl3) δ 5.69 – 5.53 (m, 1H), 4.36 (d, J = 1.3 Hz, 4H), 4.08 (ddd, J = 6.7, 4.8, 1.9 Hz, 8H), 3.67 (t, J = 6.5 Hz, 2H), 2.33 (s, 10H), 1.97 (s, 3H), 1.75 – 1.54 (m, 20H), 1.41 (d, J = 7.9 Hz, 10H)). The numeral average 6 ACS Paragon Plus Environment

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molecular weight were estimated by size exclusion chromatography (SEC), using THF as eluent with 0.5 mL/min flow rate and temperature set at 35°C. The instrument (Agilent, 1100 series, Germany) is equipped with two detector in series (ultraviolet, UV, and differential refractive index, RI) and a pre-column with PL-oligopore column (measuring range 0 – 4.500 Da). Universal calibration was applied, based on polystyrene standards (Polymer Laboratories) and using MarkHouwink parameters for PCLnMA, (K=2.00 x 10-4 [dL/g] and a=0.571[-]). 43 2.2 Synthesis of PCLnChMA Positively Charged Macromonomers Synthesis of PCLnChMA macromonomers has been done through a three steps process, as reported in Scheme 1. The first step is the synthesis of the PCLnMA macromonomers with the same procedure above described. Then, the macromonomers have been functionalized with a carboxyl group through a acylation reaction with succinic anhydride. In particular, PCLnMA (1 eq.) and succinic anhydride (1.1 eq.) were heated up in a stirred flask at 90 ± 1 °C with the temperature controlled by an external oil bath. The reaction was carried out for 18 hours. The third step involves a condensation reaction between the carboxyl group present in the acylated macromonomer and the hydroxyl group of the choline chloride. In this case, DCC and DMAP are required as carboxyl group activating agent and as nucleophilic catalyst, respectively. Briefly, to a magnetically stirred mixture of acylated PCLnMA macromonomer (1 eq), choline chloride (1.3 eq), DMAP (0.2 eq) and acetonitrile (solution 0.1 M with respect to the macromonomer), DCC (1.5 eq) was added dropwise using a siringe pump (Model NE-300, New Era Pump, US). The temperature was maintained below 5 ± 1 °C over a period of 1 hour so as to slow down the reaction and to avoid the occurrence of side reactions. At the end of the DCC addition, the ice bath was removed and the reaction was left overnight at room temperature under continuous stirring. Finally, the mixture was filtered three times to remove the dicyclohexylurea and then, acetonitrile has been removed under vacuum. In order to purify the final product, the mixture has been dissolved in chloroform and washed with a 0.5 M aqueous solution of hydrochloric acid to remove DMAP and the unreacted choline chloride 7 ACS Paragon Plus Environment


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and a brine solution in order to avoid the formation of an emulsion system due to the presence of charged monomers. Finally, produced macromonomers were all stored in dichloromethane at 4°C waiting for further use. All the positively charged functionalized macromonomers were analyzed by 1

H-NMR. As an example, for PCL3ChMA, 1H NMR (400 MHz, DMSO) δ 6.02 (s, 1H), 4.47 (d, J =

2.0 Hz, 2H), 4.34 – 4.21 (m, 4H), 4.06 – 3.90 (m, 6H), 3.74 – 3.57 (m, 2H), 3.13 (d, J = 4.3 Hz, 9H), 2.69 – 2.56 (m, 4H), 2.37 – 2.19 (m, 6H), 1.88 (s, 3H), 1.64 – 1.42 (m, 12H), 1.39 – 1.20 (m, 6H). 2.3 Nanoparticle Synthesis In order to obtain positively charged biodegradable NPs, BEP and MSSEP have been used as described in the literature.

41, 44

In the first case, the monomer was put in the reactor together with

the surfmer agent and deionized water, while in the second one, the monomer was slowly added into the system using a syringe pump with a variable flow rate. In both cases, the reactions are performed under nitrogen atmosphere, keeping constant the speed of the magnetic stirring and the temperature due to the external oil bath set at 80± 1°C. The reactions were carried out for 2 hours. In addition, PEGMA has been used in all of these synthesis as a steric co-surfmer to increase the stability of the NP latex, as already reported in the literature.

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In a typical experiment, 50 ml of

distilled water have been used with a total monomer concentration equal to 2% w. Different Surfmer/Monomer (S/M) weight ratios have been chosen to tune several dimensions (i.e. S/M = 2, 5, 8, 10, 15% WPCLnChMA/WPCLnMA). Dynamic laser light scattering (DLLS, Zetasizer Nano Series, Malvern Instruments) allows the evaluation of NP size, polidispersity index (PDI) and ζ-potential. In particular the PSD is estimated using the first- and second- order cumulates of the light scattered intensity as reported elsewhere. 45 2.4 Degradable Behaviour


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NPs with different PCLnMA macromonomer and PCLnChMA surfmer co-polymerized with PEGMA have been selected for degradation studies. 1 mL of NP dispersion (polymer/water ratio of 2%, w/w) was dissolved in 1 mL of aqueous medium and maintained in a heating block at 37.0 ± 0.1 °C. Another sample was dissolved in 1 mL of deionized water and put in the oven at 90.0 ± 1 °C. The evolution of both size, PDI and relative scattering intensity of the samples was measured through DLLS until complete polymer degradation. DLLS measurements were performed out using 137° backscatter angle. All the experimental data are an average value of five measurements of the same sample.

3. Results and Discussions It is important to well characterize the obtained NPs in term of size, charge and degradation behaviour in order to understand their use in further applications. For instance, a highly positive charge on the NP surface allows a better separation in the flocculation processes or guarantees a high drug loading of negatively charge drug in biomedical field. Also NP size obviously influences most of the processes in which they are employed. For example, in the flocculation processes, NP size influence the sedimentation rate of the flocs. Finally, the degradation of these NPs was evaluated in two different environments and temperatures to prove the NP suitability for both biomedical and industrial applications. 3.1 Characterization of PCLnMA and Positively Charged PCLnChMA Macromonomers PCLnMA and PCLnChMA were firstly characterized via 1H-NMR to evaluate the real number of the chain (n) (Figure 1) and the efficiency of functionalization achieved (Figure 2). In addition, for PCLnMA, the uncharged macromonomer, the number average molecular weight (Mn) was estimated by SEC. The results are shown in the Table 1 and Figure 3. Table 1 shows the comparison, in term of chain length and molecular weight, among the theoretical values and the real ones coming from 1H-NMR and SEC, respectively. 9 ACS Paragon Plus Environment


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Table 1: Experimental characterizations of the synthesized macromonomers 1

Theoretical

H-NMR

SEC

Macromonomer n

Mn [Da]

n

Mn [Da]

Mn [Da]

PCL2MA

2

358

2.7

438

395

PCL3MA

3

473

3.55

529

475

PCL5MA

5

701

5.329

734

730

Figure 1: 1H-NMR of PCLnMA in CDCl3


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Figure 2: 1H-NMR of the PCLnChMA in DMSO-d6 From Figure 1, it is possible to estimate the length of the chain (n) after the peaks integration. According to the letters used in the molecular structure, C is representative of the total number of repeat ε-caprolactone units added, while D is the total number of terminal groups. As a result, it is possible to evaluate the chain length, n, by apply the formula: n=

C +1 D

in which C and D are the value of the integrals, assuming as a reference the peak of the hydrogen in the double bond. All the MW data are reported in Table 1, in which there is also a comparison among the theoretical and the experimental results. The difference between the results obtained by SEC and NMR is due to the polystyrene (PS) calibration used for the evaluation of the molecular weight from the SEC. Figure 2 confirmed the achieved functionalization, due to the presence of the peaks of the succinic anydride and choline chloride, consequently the disappearance of the peak at 3.67 ppm.



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Figure 3: SEC traces from the three synthesized macromonomers PCLnMA. The SEC trace of HEMA is reported to identify the first peak on the right

Figure 3 shows the SEC chromatograms of the three PCLnMA macromonomers. The wavy behaviour on the right is related to the ability of SEC to efficiently separate the oligomers during the analysis. Nevertheless, the shifting trend of the peaks confirms the increase of the molecular weight for the three synthesized macromonomers. It is important to note that the peaks are not centered on the theoretical chain length but are slightly shifted on the left, coherently with the molecular weight values obtained by NMR. This behavior is probably due to the uncomplete conversion of HEMA in the first step of the synthesis. 12 ACS Paragon Plus Environment

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3.2 PCLnChMA based NP Once positively charged functionalized macromonomers were synthesized, they have been employed as surfmers for the synthesis of positively charged NPs. At first, the production of PCLbased NPs was carried out using PCLnChMA, as a cationic surfmer and PEGMA as a steric one. In the Tables S1 and S2, it is possible to appreciate the differences in term of size, ζ-potential and polydispersity of NPs synthesized through BEP and MSSEP conditions. In this way it is possible to obtain a wider range of NP size due to the use of two polymerization techniques that differs for the homogeneous nucleation.46 In particular, in BEP, where the total amount of monomer is present since the beginning of the reaction part of the surfactant is adsorbed on the monomer droplets and is not involved in the particle nucleation. On the other hand, when starved semi-batch conditions are established, the monomer droplets are not present during all the process and smaller NP size can be obtained. Nevertheless the nucleation, which is active for the whole polymerization processes in MSSEP condition, leads to a slightly increase of the PDI as shown in Table S2. During the polymerization the larger the amount of surfmer the smaller are the NPs produced, whereas the surface charge increases. Stable and monodispersed NPs with PDI values lower than 0.2 were obtained in all cases. It is worth to note that BEP leads to larger NPs compared to MSSEP according to the literature.47 By changing the S/M ratio, NPs size are comprised between 80 nm and 115 nm for BEP and between 63 nm and 75 nm for MSSEP. DLLS measurements also demonstrate that NPs have positive ζ-potential values comprised between + 34 and +43 mV. It is worth to note that by increasing S/M weight ratio, the ζ-potential increases in both the polymerization techniques used. The reason is the increase of the amount of the positively charged surfmer (PCLnChMA), keeping constant the quantity of PEGMA (PEGMA/M=20%). PEGMA is necessary to ensure the stability of the NPs. Two different PEGMA have been adopted leading to a decrease in the

ζ−potential from PEGMA1000 to PEGMA2000 (Table 2). The results clearly indicate that NPs synthesized using PEGMA1000 are unstable in a biological environment and cannot be considered 13 ACS Paragon Plus Environment


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suitable for biomedical applications. NP instability can be ascribed to the presence of proteins and other negatively charged compounds in the growth medium that can screen the positive charges on the NP surface, promoting the aggregation of the NP latexes. Consequently, the more efficient PEGMA2000 is used instead of PEGMA1000 for NP synthesis to enhance the colloidal stability of the NP latexes. The effect of the surface charge in the two different cases is evaluated as well. PEGMA2000 chains are longer than the PEGMA1000 chains and screen the positive charges more efficiently. This hypothesis is confirmed by the ζ-potential values of two NPs that were synthesized using the same amount of the PCL2ChMA macromonomer but with PEGMAs of two different molecular weights (i.e., PEGMA1000 and PEGMA2000). The PEGMAm/M percentage weight ratio was constant at 20%. Table 2: Comparison of NP z-potential obtained using PEGMA at two different molecular weight Macromonomer Surfmer

S/M

PEGMAm/M

ζ-POT

[w/w]

[w/w]

[mV]

PCL2MA

PCL2ChMA

5%

20% (m=1000)

+51

PCL2MA

PCL2ChMA

5%

20 % (m=2000)

+38

It is noteworthy that the ζ-potential, although lower when PEGMA2000 was used in place of PEGMA1000, was still positive; consequently, NPs synthesized using PEGMA2000 were suitable for the delivery of negatively charged compounds such as siRNA. Respect to the Table S2, Table S3 shows the properties of the NPs synthesized through MSSEP using both the positively charged macromonomer and the unfuctionalized one, changing the length of the CL units. It means a better understanding of the influence of the CLn on the final NPs. The NP size decreases with the CL units, while the surface charge is approximately constant as the relative amount of charged macromonomer is the same in each synthesis. 14 ACS Paragon Plus Environment

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Afterwards, positively charged NPs have been synthesized through BEP using three different PCLnChMA (n=2, 3, 5) and the PEGMA as steric stabilizer, as shown in Table S4. The aim is to increase the positively charge on the NP surface so as to improve the property of the obtained products. It means a higher amount of loaded siRNA in the case of gene therapy, an increasing efficiency in the flocculation processes and a higher viscous system in the flooding applications. In fact, the ζ-potential values are higher than the previous results. It can be seen that the NP size decreases with the PCL chain length. This is due to the increase of the hydrophobicity of the longer PCL chains since more hydrophobic NPs are generally less swelled. The surface charge trend is opposite because of the higher molar concentration of positively charges involved in the reaction. 3.3 Degradation study of cationic NPs Two different PCL-based cationic NPs have been selected for further degradation studies in order to underline their degradability. The purpose is to investigate the effect of the PCL chain length (n) on the NP degradation process. The chosen NPs are characterized by n values equal to 2 and 3 (both for monomer and cationic surfmer). In particular, in these experiments the degradation kinetics in cell medium at 37°C and in distilled water at 90°C have been deeply characterized. The first one is necessary to state the degradation in biomedical applications, whereas the second is chosen to simulate the degradation due to the hydrolysis in industrial processes. In particular, cell medium is a biological relevant environment since it is used to grow cells in in vitro experiments and it contains proteins that are also present in the blood and that act as surfactants for the oligomers that are released form the NPs during the degradation. This phenomenon increases the degradation rate in biological environment, as already reported in literature.

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On the other hand, distilled water is

adopted to evaluate the hydrolysis due to the simple aqueous environments. In those systems, according to prior studies, degradation is based on a bulk erosion process, that occurs through the hydrolysis of ester bonds.39 The degradation reaction proceeds in this way in both PCL-based and PCLCh-based chains and the subsequent release of PCL-based oligomers which can be further 15 ACS Paragon Plus Environment


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hydrolyzed to 6-hydroxycaproic acid, succinic acid, choline moieties, water soluble poly(HEMA) chains that can degrade to poly(methacrylic acid) and ethylene glycol. 16, 43, 48 (Scheme 2)

Scheme 2: Degradation pathway of synthesized NPs

PCL2MA/PCL2ChMA and PCL3MA/PCL3ChMA NPs have been monitored by studying the evolution of the PSD during the time in cell medium (Figure 4) and in water (Figure 5). Further results obtained from the DLS analysis (Figure S1 and S2) and the pH (Figure S3) over the time can be found in the the Supporting Information, in which also the cell viability before and after the hydrolysis are reported (Figure S4).


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Figure 4: Evolution of PSD for PCL2MA/PCL2ChMA (a) and PCL3MA/PCL3ChMA (b) during degradation in cell medium at 37°C over the time.

Figure 5: Evolution of PSD for PCL2MA/PCL2ChMA (a) and PCL3MA/PCL3ChMA (b) during degradation in distilled water at 90°C over the time.

The lipophilic nature influences the degradation time of the NPs, showing that PCL3-based NPs require a higher number of hydrolytic steps rather than PCL2-based ones. In addition, the degradation rate increases with temperature, as expected. At the beginning of the degradation study only a single peak is observed which underlines the monodisperse distribution of the NPs. Firstly, the polyester chains are continuously swollen and 17 ACS Paragon Plus Environment


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hence the NPs increase the hydrophilicity until the swelling mechanism reaches an equilibrium state. Afterwards, the hydrolysis process begins to strongly affect the structure of the particle, increasing particle dimension due to the breakage of CL-based chains, till the formation of a watersoluble polymer dissolved in the aqueous medium (verified by a low value of scattering light intensity, Figure S1). Regarding the degradation at 90°C in water, it is reasonable to suppose that certain extent of particle aggregation of NPs occurs due to the high temperature. In this case the evaluation of NP degradation by the scattering intensity may be not accurate (Figure S2). For this reason, Figure S3 shows the evolution of pH over the time at the same temperature. As can be seen, the dynamic of pH is similar to the dynamic of scattering intensity, so it is possible to state that NP degradation occurs in about 5-6 h at 90°C. 4. Conclusions In this work, cationic polyester monomers are synthesized throughout a three step functionalization, that involved ring opening polymerization, acylation with succinic anhydride and the condensation reaction with choline chloride. The obtained materials, having different length of CL units, are used to produce NPs via MSSEP and BEP processes. The same NPs have been studied in order to understand their degradation behaviour. It is possible to summarize that for PCL-based NPs the macromonomer chain length is the key parameter that directly influences the degradation of the NPs and a wide range of degradation rates can be obtained tuning the number of CL units in the polymeric structure. Therefore, several NPs may be obtained in order to satisfy a specific range of degradation time (i.e. retention time inside the human body) suitable for biomedical and industrial applications. 5. Supporting Information Details related to the characterization of the obtained NPs are reported in the Supporting Information. 18 ACS Paragon Plus Environment

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6. Bibliography (1)

Matyjaszewski, K., Cationic Polymerizations: Mechanisms, Synthesis & Applications. CRC Press: 1996. (2) Egorova, K. S.; Seitkalieva, M. M.; Posvyatenko, A. V.; Khrustalev, V. N.; Ananikov, V. P., Cytotoxic activity of salicylic acid-containing drug models with ionic and covalent binding. ACS Med. Chem. Lett. 2015, 6, 1099-1104. (3) Arshady, R., Suspension, emulsion, and dispersion polymerization: a methodological survey. Colloid Polym. Sci. 1992, 270, 717-732. (4) Lovell, P. A.; El-Aasser, M. S., Emulsion polymerization and emulsion polymers. Wiley: 1997. (5) Eliseeva, V. I.; Ivanchev, S.; Kuchanov, S.; Lebedev, A., Emulsion polymerization and its applications in industry. Springer Science & Business Media: 2012. (6) Roy, K.; Mao, H.-Q.; Huang, S.-K.; Leong, K. W., Oral gene delivery with chitosan–DNA nanoparticles generates immunologic protection in a murine model of peanut allergy. Nat. Med. 1999, 5, 387-391. (7) Jain, S.; Kumar, D.; Swarnakar, N. K.; Thanki, K., Polyelectrolyte stabilized multilayered liposomes for oral delivery of paclitaxel. Biomaterials 2012, 33, 6758-6768. (8) Schulz, J. D.; Gauthier, M. A.; Leroux, J.-C., Improving oral drug bioavailability with polycations? Eur. J. Pharm. Biopharm. 2015, 97, 427-437. (9) Nayerossadat, N.; Maedeh, T.; Ali, P. A., Viral and nonviral delivery systems for gene delivery. Adv. Biomed. Res. 2012, 1, 27. (10) Ramamoorth, M.; Narvekar, A., Non viral vectors in gene therapy-an overview. J Clin Diagn Res 2015, 9, GE01-GE06. (11) Luten, J.; van Nostrum, C. F.; De Smedt, S. C.; Hennink, W. E., Biodegradable polymers as non-viral carriers for plasmid DNA delivery. J. Controlled Release 2008, 126, 97-110. (12) Tranchant, I.; Thompson, B.; Nicolazzi, C.; Mignet, N.; Scherman, D., Physicochemical optimisation of plasmid delivery by cationic lipids. J Gene Med 2004, 6, S24-S35. (13) Han, S.; Wan, H.; Lin, D.; Guo, S.; Dong, H.; Zhang, J.; Deng, L.; Liu, R.; Tang, H.; Dong, A., Contribution of hydrophobic/hydrophilic modification on cationic chains of poly (ε-caprolactone)-graft-poly (dimethylamino ethylmethacrylate) amphiphilic co-polymer in gene delivery. Acta Biomater. 2014, 10, 670679. (14) Wurtmann, E. J.; Wolin, S. L., RNA under attack: cellular handling of RNA damage. Crit. Rev. Biochem. Mol. Biol. 2009, 44, 34-49. (15) Yang, N. J.; Hinner, M. J., Getting across the cell membrane: an overview for small molecules, peptides, and proteins. Site-Specific Protein Labeling: Methods and Protocols 2015, 29-53. (16) Dragoni, L.; Ferrari, R.; Lupi, M.; Cesana, A.; Falcetta, F.; Ubezio, P.; D’Incalci, M.; Morbidelli, M.; Moscatelli, D., Small interfering RNA delivery through positively charged polymer nanoparticles. Nanotechnology 2016, 27, 125102. (17) Tanaka, K.; Takeda, H.; Kawano, M.; Miyahara, I., Cationic polymer flocculant prepared by reacting epichlorohydrin and a Mannich reaction product. In Google Patents: 1979. (18) Bratby, J., Coagulation and flocculation in water and wastewater treatment. IWA publishing: 2006. (19) Cadotte, M.; Tellier, M.-E.; Blanco, A.; Fuente, E.; Van De Ven, T. G.; Paris, P., Flocculation, retention and drainage in papermaking: a comparative study of polymeric additives. Can. J. Chem. Eng. 2007, 85, 240240. (20) Zhong, J.; Sun, X.; Wang, C., Treatment of oily wastewater produced from refinery processes using flocculation and ceramic membrane filtration. Sep. Purif. Technol. 2003, 32, 93-98. (21) Lee, C. S.; Robinson, J.; Chong, M. F., A review on application of flocculants in wastewater treatment. Process Saf. Environ. Prot. 2014, 92, 489-508. (22) González, G.; José, C.; Asua, J. M., Synthesis of cationic flocculants in continuous reactors. Chem. Eng. J. 2010, 162, 753-759. (23) Lu, L.; Pan, Z.; Hao, N.; Peng, W., A novel acrylamide-free flocculant and its application for sludge dewatering. Water Res. 2014, 57, 304-312. 19 ACS Paragon Plus Environment


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Page 20 of 24

(24) Rooney, T. R.; Gumfekar, S. P.; Soares, J. B.; Hutchinson, R. A., Cationic Hydrolytically Degradable Flocculants with Enhanced Water Recovery for Oil Sands Tailings Remediation. Macromol. Mater. Eng. 2016, 301, 1248-1254. (25) Tzoupanos, N.; Zouboulis, A. In Coagulation-flocculation processes in water/wastewater treatment: the application of new generation of chemical reagents, 6th IASME/WSEAS international conference on heat transfer, thermal engineering and environment (HTE’08), August 20th–22nd, Rhodes, Greece, 2008; 2008; pp 309-317. (26) Gao, C.; Shi, J.; Zhao, F., Successful polymer flooding and surfactant-polymer flooding projects at Shengli Oilfield from 1992 to 2012. J. Pet. Explor. Prod. Technol. 2014, 4, 1-8. (27) Zhao, X.; Liu, L.; Wang, Y.; Dai, H.; Wang, D.; Cai, H., Influences of partially hydrolyzed polyacrylamide (HPAM) residue on the flocculation behavior of oily wastewater produced from polymer flooding. Sep. Purif. Technol. 2008, 62, 199-204. (28) Solberg, D.; Wågberg, L., Adsorption and flocculation behavior of cationic polyacrylamide and colloidal silica. Colloids Surf., A 2003, 219, 161-172. (29) Chen, H.-x.; Tang, H.-m.; Gong, X.-p.; Wang, J.-j.; Liu, Y.-g.; Duan, M.; Zhao, F., Effect of partially hydrolyzed polyacrylamide on emulsification stability of wastewater produced from polymer flooding. J. Pet. Sci. Eng. 2015, 133, 431-439. (30) Zegeye, E.; Vermuë, M.; Kleinegris, D.; Eppink, M.; Wijffels, R.; Olivieri, G., Dosage effect of cationic polymers on the flocculation efficiency of the marine microalga Neochloris oleoabundans. Bioresour. Technol. 2015, 198, 797-802. (31) Song, B.; Hu, X.; Shui, X.; Cui, Z.; Wang, Z., A new type of renewable surfactants for enhanced oil recovery: dialkylpolyoxyethylene ether methyl carboxyl betaines. Colloids Surf., A 2016, 489, 433-440. (32) Jamaly, S.; Giwa, A.; Hasan, S. W., Recent improvements in oily wastewater treatment: Progress, challenges, and future opportunities. J. Environ. Sci. 2015, 37, 15-30. (33) Chen, Z.; Zhao, X.; Wang, Z.; Fu, M., A comparative study of inorganic alkaline/polymer flooding and organic alkaline/polymer flooding for enhanced heavy oil recovery. Colloids Surf., A 2015, 469, 150-157. (34) Sauer, R.; Froimowicz, P.; Schöller, K.; Cramer, J. M.; Ritz, S.; Mailänder, V.; Landfester, K., Design, Synthesis, and miniemulsion polymerization of new phosphonate surfmers and application studies of the resulting nanoparticles as model systems for biomimetic mineralization and cellular uptake. Chem. - Eur. J. 2012, 18, 5201-5212. (35) Fischer, V.; Landfester, K.; Munoz-Espi, R., Molecularly controlled coagulation of carboxylfunctionalized nanoparticles prepared by surfactant-free miniemulsion polymerization. ACS Macro Lett. 2012, 1, 1371-1374. (36) Gary, D. J.; Puri, N.; Won, Y.-Y., Polymer-based siRNA delivery: perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery. J. Controlled Release 2007, 121, 64-73. (37) Xue, Y.; Xiao, H.; Zhang, Y., Antimicrobial polymeric materials with quaternary ammonium and phosphonium salts. Int. J. Mol. Sci. 2015, 16, 3626-3655. (38) Ferrari, R.; Colombo, C.; Casali, C.; Lupi, M.; Ubezio, P.; Falcetta, F.; D’Incalci, M.; Morbidelli, M.; Moscatelli, D., Synthesis of surfactant free PCL–PEG brushed nanoparticles with tunable degradation kinetics. Int. J. Pharm. 2013, 453, 551-559. (39) Ferrari, R.; Colombo, C.; Dossi, M.; Moscatelli, D., Tunable PLGA-Based Nanoparticles Synthesized Through Free-Radical Polymerization. Macromol. Mater. Eng. 2013, 298, 730-739. (40) Yu, Y.; Ferrari, R.; Lattuada, M.; Storti, G.; Morbidelli, M.; Moscatelli, D., PLA-based nanoparticles with tunable hydrophobicity and degradation kinetics. J. Polym. Sci., Part A: Polym. Chem. 2012, 50, 51915200. (41) Ferrari, R.; Yu, Y.; Lattuada, M.; Storti, G.; Morbidelli, M.; Moscatelli, D., Controlled PEGylation of PLA-Based Nanoparticles. Macromolecular Chemistry and Physics 2012, 213. (42) Ferrari, R.; Cingolani, A.; Moscatelli, D., Solvent Effect in PLA-PEG Based Nanoparticles Synthesis through Surfactant Free Polymerization. Macromol Symp 2013, 324, 107-113. (43) Ferrari, R.; Yu, Y.; Morbidelli, M.; Hutchinson, R. A.; Moscatelli, D., ε-Caprolactone-based macromonomers suitable for biodegradable nanoparticles synthesis through free radical polymerization. Macromolecules 2011, 44, 9205-9212. 20 ACS Paragon Plus Environment

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(44) Chen, Y.; Sajjadi, S., Particle formation and growth in ab initio emulsifier-free emulsion polymerisation under monomer-starved conditions. Polymer 2009, 50, 357-365. (45) Perevyazko, I.; Vollrath, A.; Hornig, S.; Pavlov, G. M.; Schubert, U. S., Characterization of poly (methyl methacrylate) nanoparticles prepared by nanoprecipitation using analytical ultracentrifugation, dynamic light scattering, and scanning electron microscopy. J. Polym. Sci., Part A: Polym. Chem. 2010, 48, 3924-3931. (46) Dossi, M.; Ferrari, R.; Dragoni, L.; Martignoni, C.; Gaetani, P.; D'Incalci, M.; Morbidelli, M.; Moscatelli, D., Synthesis of Fluorescent PMMA-Based Nanoparticles. Macromol. Mater. Eng. 2013, 298, 771-778. (47) Sajjadi, S., Nanoparticle formation by monomer-starved semibatch emulsion polymerization. Langmuir 2007, 23, 1018-1024. (48) Colombo, C.; Dragoni, L.; Gatti, S.; Pesce, R. M.; Rooney, T. R.; Mavroudakis, E.; Ferrari, R.; Moscatelli, D., Tunable Degradation Behavior of PEGylated Polyester-Based Nanoparticles Obtained Through Emulsion Free Radical Polymerization. Ind. Eng. Chem. Res. 2014, 53, 9128-9135.



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Azzurra Agostini received the MSc in Chemical Engineering cum laude at Politecnico di Milano in 2015. She is attending PhD in industrial Chemistry and Chemical Engineering at Politecnico di Milano. Her research is focused on the synthesis of polymer nanoparticles for oral delivery and tissue engineering.

Alberto Cesana received the MSc in Chemical Engineering cum laude at Politecnico di Milano in 2015. After graduation, he worked as a temporary researcher for 1 year at Politecnico di Milano; since November 2016 he is employed in SICAD, an Italian company manufacturing pressure sensitive adhesive, as a member of R&D board.

Simone Gatti received the MSc in Chemical Engineering at Politecnico di Milano in 2013. He is attending PhD in industrial Chemistry and Chemical Engineering at Politecnico di Milano. His research is focused on the development of polymeric nanoparticles and polymer drug conjugates for drug delivery applications.


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Davide Moscatelli received the MSc in Chemical Engineering cum laude at Politecnico di Milano in 2002 and his PhD in 2006. He is Associate Professor in Applied Physical Chemistry at the Department of Chemistry, Materials and Chemical Engineering at Politecnico di Milano. His main research interests concern the application of physical chemistry fundamentals to analyze industrial processes of chemical nature, the materials synthesis (organic, inorganic and polymers) and their environmental and safety aspects. The applications are related to industrial processes with chemical implications, polymers and inorganic materials for advanced technologies, and nanotechnology (primarily nanoparticle synthesis). Davide Moscatelli is co-author of more than 100 papers, 5 national and international patents. He is cofounder of Captive Systems srl, a spin-off company of Politecnico di Milano.



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TOC 326x152mm (96 x 96 DPI)

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Synthesis and Degradation Study of Cationic Polycaprolactone-Based

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