1 Molecules 2014, 19, 1-x manuscripts; doi:10.

3390/molecules190x0000x

2 OPEN ACCESS

3 molecules
4 ISSN 1420-3049
5 www.mdpi.com/journal/molecules
6 Article

7 Effect of pH and Monomer Dosing Rate in the Anionic

8 Polymerization of Ethyl Cyanoacrylate in Semicontinuous
9 Operation
10 H. Saade 1,*, S. Torres 1, 2, C. Barrera 1, J. Sánchez 1, Y. Garza 2, R.G. López 1
11 1 Departamento de Procesos de Polimerización, Centro de Investigación en Química Aplicada, Blvd.

12 Enrique Reyna No. 140, C. P. 25294, Saltillo, Coahuila, México.

13 2 Departamento de Biotecnología, Facultad de Ciencias Químicas, Universidad Autónoma de

14 Coahuila, Blvd. V. Carranza y José Cárdenas Valdés 25280, Saltillo, Coahuila, México.

15 * Author to whom correspondence should be addressed; E-Mail: hened.saade@ciqa.edu.mx;

16 Tel: +52-844-4389-847; Fax: +52-844-4389-839

17 Received: / Accepted: / Published:

18
19

20 Abstract: Nanoparticles of poly(ethyl cyanocrylate) with more than 10 % solids content

21 were prepared by semicontinuous heterophase polymerization at monomer starved
22 conditions varying the initial pH in the interval 1 to 1.75 and at two monomer dosing rates.
23 Measurements by scanning-transmission electronic microscopy allowed to identify an
24 inverse dependence of particle size on pH. Furthermore, all the polymerizations conducted
25 at the slower monomer dosing rate rendered two particle populations, with the larger one
26 formed from the aggregation of a fraction of the smaller particles. It was believed that the
27 so slow addition of the monomer caused the formation of very small but instable particles,
28 thereby a fraction of which aggregated to reduce the total interface particles-aqueous phase
29 and increase the latex stability. An increase in the monomer dosing rate leaded to larger
30 and more stable particles in such way that only one population of nanoparticles with
31 around 40 nm in average diameter was obtained.

32 Keywords: poly(ethyl cyano acrylate) latexes; high solid content; poly(ethyl cyano
33 acrylate) nanoparticles sub-50 nm.
34

35
 Molecules 2014, 19 2

1 1. Introduction

2 The great potential of drug delivery nanosystems for enhancing the efficacy of drugs and to reduce
3 their side effects has generated enormous interest in the study of polymeric nanoparticles as basis for
4 the development of such systems [1, 2]. Poly(alkyl cyanoacrylate), PACA, nanoparticles are a very
5 interesting material for the design of this type of systems due to their well-known biocompatibility and
6 biodegradability [3, 4]. Since Couvreur et al. reported the anionic polymerization of methyl
7 cyanoacrylate (MCA) and ethyl cyanoacrylate (ECA) in emulsion [5], a number of research groups
8 have documented studies on the preparation of nanoparticles and loaded them with different drugs [3,
9 4]. In accordance with the reports on the subject, polymerization in an aqueous media stabilized with
10 an amphiphilic is the most widely used technique for preparing PACA nanoparticles [3, 4].
11 Agree with the specialized literature, type and surfactant concentration [6-11] and initial pH [7-9,
12 12-15] are the most studied variables in the PACA nanoparticles preparation. All of these works
13 emphasize the influence of these variables on particle size, mainly due to the possible use of drug-
14 loaded PACA nanoparticles in the development of drug delivery systems, in which ultrafine
15 nanoparticles are desirable. Such particles, ranging 10-50 nm in average diameter, are very attractive
16 due to their smallness, which undermines their clearance by the immunological system [16]. An
17 additional advantage of the drug-loaded nanoparticles with diameters smaller than 50 nm is the
18 possibility they offer to cross through intestinal walls entering the bloodstream [17]. The reports in
19 literature indicate that different types of dextrans [6, 10, 12-14] and Tweens [5, 6, 11, 12] are the most
20 evaluated surfactants in the emulsion polymerization for PACA obtaining and that in general the
21 smaller sizes are obtained when Tweens were used. A revision of the quoted reports indicates that
22 average diameters as large as 400 nm were obtained by Beham et al. [13] who used Dextran 70 in
23 emulsion polymerization of butyl cyanoacrylate (BCA) at different pH´s between 2 and 3. In contrast,
24 Douglas et al. reported the obtaining of poly butyl cyanoacrylate (PBCA) nanoparticles with around 50
25 nm in average diameter when using Tween 60 in an emulsion polymerization at pH 2.25 [6]; in fact,
26 they obtained larger particles when tested Tweens with lower molecular weight (TW20 and TW40),
27 which suggested an inverse relationship between particle size and Tween molecular weight. On the
28 other hand, the reports on pH influence have demonstrated that polymerizations carried out at pH´s
29 lower than 1 and higher than 3.5 produce very large particles and that smaller particles are obtained at
30 pH´s close to 2 [12, 13]. It is noticeable that practically all the works reporting particle sizes are based
31 on measurements by light scattering, while only a few include electronic microscope determinations
32 [5, 9]; however, the latter do not show particle size distributions from micrographs, but use these
33 results as complement of light scattering measurements. Nevertheless, the report of Yang et al. [9]
34 calls attention due the very small particle sizes of PBCA they said have obtained. These authors
35 documented the results of emulsion polymerization of BCA, showing micrographs with two particle
36 populations, one showing diameters smaller than 50 nm and another with much larger sizes. It would
37 have been interesting to know about the fraction of the particle populations in the latexes, however
38 taking into account the turbidity of the dispersions mentioned by the authors, surely larger particles
39 constituted the largest fraction.
40 The aim of the study presented herein was to know how the use of a Tween with a higher molecular
41 weight (Tween 80, or TW80) than those used in the works reported in the literature affect the particle
 Molecules 2014, 19 3

1 size, in which determination the electronic microscopy technique was employed. In this study
2 polymerizations at different pH´s between 1 and 1.75 were carried out in a semicontinuous manner,
3 that is to say, dosing the monomer ECA over a micellar solution. This monomer adding policy has
4 been used successfully by our group under the name of semicontinuous heterophase polymerization at
5 monomer starved conditions to obtain polymeric nanoparticles with average diameters substantially
6 smaller than 50 nm [19-21]. Despite those polymerizations proceeded via free radical mechanism, it
7 was expected that this technique also would lead to very small particles, taking into account that the
8 anionic polymerization of ECA also would take place in surfactant stabilized compartments.
9

10 2. Results and Discussion

11 All the polymerizations practically attained total monomer conversion, which resulted in latexes
12 with final solid contents close to 10.5 %; this looks interesting due to in accordance with the
13 specialized literature solid contents no higher than 5-6 % are common in the PACA latexes obtained
14 by emulsion polymerization [6-12, 15]. It is pertinent to make clear that practically none of the quoted
15 reports mentions monomer conversion values, so the corresponding solid contents were estimated from
16 the reported formulations and assuming total monomer conversion.
17 Figures 1 includes a representative micrograph of a sample of the final latex prepared at pH 1,
18 where is evident the existence of two particle populations. This figure also includes the histogram of
19 particle diameters for each one of the populations, which were constructed from the measurement of
20 the size of a great number of particles in the set of micrographs. Figures 2 and 3 show the micrographs
21 and the corresponding histograms for the final latexes prepared at pH 1.5 and 1.75, respectively. On
22 the other hand, Table 1 shows the values for Dn, polydispersity index and the number of particles
23 measured to obtain those values, corresponding to each one of the populations in the prepared latexes.
24 From the micrographs in Figures 1 to 3 became evident that the large particles are formed by the
25 aggregation of a fraction of the smaller ones and that the individuality of the latter is preserved, that is
26 to say, that they do not coalesce. Additionally, an inspection of data in Table 1 indicates that the
27 average size of both populations decreases as pH values increases and that the small particle
28 populations are formed of very small particles, 22.0 and 11.9 nm in Dn for polymerizations at pH 1 and
29 1.75, respectively. Values as small than these, determined by electronic microscopy have not been
30 previously reported in the literature. The questions that arise now are why such small particles are
31 formed, why the smaller ones are obtained at the higher pH and why a fraction of the smaller particles
32 aggregates to give rise larger ones in all polymerizations.

33

34

35

36

37
 Molecules 2014, 19 4

1 Figure 1. Micrograph and histograms of a sample taken at the end of the polymerization at
2 pH 1.0.

4
5

6 Figure 2. Micrograph and histograms of a sample taken at the end of the polymerization at
7 pH 1.5.

9
10
11
12
 Molecules 2014, 19 5

1 Figure 3. Micrograph and histograms of a sample taken at the end of the polymerization at
2 pH 1.75.

4
5

6 Table 1. Results of STEM measurements for samples from polymerizations at different pH´s.
pH Population 1 Population 2
Measured Dn Dw/Dn Measured Dn Dw/Dn
particles (nm) particles (nm)
1.0 536 22.0 1.4 157 119.9 1.2
1.5 774 14.4 1.4 264 67.3 1.3
1.75 1833 11.9 1.3 599 58.9 1.2
7
8 Respecting to the cause of the smallness of the PECA particles obtained in this study, it is believed
9 that the slow rate at which the monomer was added to the micellar solution, is the reason behind this
10 finding. This arises from the operation at semicontinuous heterophase polymerization, technique
11 developed by our group [19-21] that allows to obtain polymeric nanoparticles with diameters smaller
12 than 50 nm, solid contents up to 25-30 % and all the surfactant in the latex stabilizing the particles.
13 However, this technique requires to operate at the so-called monomer-starved conditions during the
14 monomer addition period [21]. Krackeler and Naidus [21] coined this term when they explained the
15 smaller particle sizes obtained in the emulsion polymerization of styrene carried out in semicontinuous
16 mode compared to the batch process. These authors resorted to the correlation developed for emulsion
17 polymerization by Smith and Ewart [22] for predicting the number of particles (NP) for the case-II
18 kinetics, in which NP is inversely proportional to the volume growth rate of the polymer particles
19 during nucleation period. When the particles are saturated with monomer, they grow at their maximum
20 rate; as a consequence, the particle nucleation is minimum. In semicontinuous emulsion
21 polymerization particle monomer saturation is attained by operating at the so-called monomer-flooded
 Molecules 2014, 19 6

1 conditions. In contrast the operation under monomer-starved conditions, that is to say, when monomer
2 in the particles is below the saturation concentration, slows down the particle growth rate resulting in a
3 larger number of smaller particles. These conditions are achieved by adding the monomer at very slow
4 dosing rates.
5 The inverse dependence between pH and particle size could be explained as follows. As it is well-
6 known [13] the mechanism of anionic polymerization of alkyl cyanoacrylates in an aqueous dispersion
7 in the presence of micelles includes an initiation via hydroxyl ions in the aqueous phase, which react
8 with the monomer near the swollen micelles surface, giving rise to a particle. Then, these anionic
9 radicals propagate inside these surfactant stabilized compartments until the growing chains terminate
10 by reacting with protons present in the monomer, which come from aqueous phase and monomers
11 stabilizer. As it can be seen the concentration of hydroxyl ions in the aqueous phase is determinant in
12 the rate of initiation and, as consequence, in the particle formation; this means that the higher hydroxyl
13 ions concentration, the more particles are formed. Thus, in view that hydroxyl ions concentration
14 increases as pH increases, an increase in the particle number and consequently smaller particle sizes in
15 the latexes should be expected.
16 Table 2 shows additional data concerning to the particle aggregation in the polymerizations. Here,
17 the particle surface area covered by one surfactant molecule was estimated by first calculating NP in
18 the latex using the following equation:
19
20
21
22 = (1)
23
24 where Cp is the polymer concentration in the latex in g/mL of water and, ρp, the polymer density in
25 g/mL, taken as 1.1 g/mL for PECA. The number of surfactant molecules was calculated from the
26 molecular weight of the surfactant (1310 g/mol), its amount in the formulation and assuming that all
27 the surfactant is on the particles. The values of particle surface area that one surfactant molecule
28 covers in Table 2 range from 3.1 to 5.7 nm2 and they increase as pH increases. In general, these values
29 are very high when compared with, for example, the value of 0.5 for the ionic surfactant sodium
30 dodecyl sulfate (SDS) [23], which suggests that TW80 molecules provide only a partial stabilization to
31 PECA particles. Data in Table 2 show indirectly that the number of surfactant molecules per surface
32 area decreases as pH increases, which suggests that the increasing number of hydroxyl ions in the
33 aqueous phase contributes to latex stabilization. However, due to the partial stabilization provided by
34 the surfactant molecules, a fraction of the particles aggregates to reduce the total interfacial area
35 seeking for a more stable latex. Table 2 also includes the estimated number of small particles that
36 aggregates to form a large one, which were obtained from the volume ratio of large to small particles
37 for each polymerization. Then, using these numbers and simple geometric calculations, it was obtained
38 an estimation of the reduction on the total interfacial area given by the sum of each one of the small
39 particles areas that aggregates to form a large one. The results shown in Table 2 indicate a similar
40 reduction for all polymerizations of around 83-85 %, that is to say, that the surface area of one average
 Molecules 2014, 19 7

1 large particle represents around 15-17 % of the area of all average small particles that had to aggregate
2 to form it.
3

4 Table 2. Data concerning to the particle aggregation in the polymerizations conducted at

5 different pH´s.
Particle surface Number of small Reduction in total area of
area covered by a surfactant particles that aggregates small particles after
pH molecule to form a large particle aggregation in one larger
(nm2) particle
(%)
1.0 3.1 162 84.5
1.5 4.7 4.7 82.4
1.75 5.7 5.7 83.2
6
7 Another feature that deserves to be treated is the difference in the polydisperisty index values
8 between the two populations observed in Table 1; in all cases the smaller nanoparticles populations
9 show a broad distribution, while those corresponding to the larger particles are consistently less broad.
10 The broad distribution are usually related to continuous particle nucleation along the reactions, due to
11 the great difference between the sizes of the particles formed early in the polymerization, who grow
12 along all the time and those nucleated at the final stages, with a reduced growth period. Continuous
13 nucleation is common in semicontinuous heterophase polymerization at monomer starved conditions
14 [18-20] under which the polymerizations in this study were conducted, and it is consequence of the
15 very low monomer concentration in the particles that prevails along the reactions. On the other hand,
16 the less broad distributions of larger particles populations suggests that another mechanism to form
17 particles is acting, in this case, the already mentioned smaller particles aggregation. Furthermore, an
18 heterogeneous aggregation, that is to say, small with large particles that belongs to the smaller particles
19 population should occur to give rise a less width particle distribution.
20 To acquire some knowledge about how the particle size evolves along the polymerizations another
21 reaction at pH 1.75 was conducted, taking a sample at 35 min from the beginning of monomer dosing
22 and another one at the end of the polymerization. The sample taken early in the reaction showed a
23 global conversion of 11.1 %, while that taken at the end of the polymerization gave a value close to
24 100 %. Figures 4 and 5 include representative micrographs of the 35 min and final samples,
25 respectively, in which can be seen two particles populations. As in the samples obtained at the end of
26 the polymerizations at the different pH´s the larger particles are formed by the aggregation of a
27 fraction of the smaller ones, which preserve their individuality. Additionally, the corresponding
28 histograms of particle diameters for each population were included in those figures. Table 3 shows Dn,
29 polydispersity index and the number of particles measured to obtain these values for the same samples.
30 The average sizes of the smaller particles population in Table 3 indicate that the particles formed at
31 early stages in the polymerizations are smaller than those obtained at the final stage. This means that
32 the smaller particles grow as the polymerization evolves probably through the recruiting of anionic
33 radicals formed near their surface by the reaction between the hydroxyl ions and the monomer
 Molecules 2014, 19 8

1 molecules which finally results in a new polymer chain inside the particle. Following the same
2 procedure used in the analysis of the data obtained in the polymerizations conducted at different pH´s,
3 it was found that around 661 smaller particles should aggregate to form one large particle at 35 min
4 from the reaction beginning. In this case, the surface area of the average large particle represents
5 around 11 % of the sum of the area of all the average small particles that had to aggregate to generate
6 it. At the end of the polymerization, the number of average small particles that aggregates to give rise
7 one average large particle is only around 108 and the surface area of the large particle is now
8 equivalent to approximately 17 % of the sum of the area of all the small particles before aggregation.
9 In general, the results obtained at the end of both polymerizations carried out at pH 1.75 show good
10 reproducibility. The differences between the results at 35 min from the reaction beginning and those at
11 the end, could be due to the very small size of the particles at the early stage of the polymerization that
12 makes them more unstable, promoting their aggregation in a number enough to more reduce their total
13 area thereby increasing the latex stability.
14

15 Figure 4. Micrograph and histograms of a sample taken at 35 min from the beginning of
16 the polymerization at pH 1.75.

17

18
19
20
21
22
23
24
25 25
26
Frequency

20
27
15
28
10
Number

29
30 5

31 0
20 40 60 80 100 120 140 160 180
32 D (nm)
33

34 Table 3. Results of STEM measurements for samples taken from polymerization at pH 1.75
Sampling time Population 1 Population 2
Measured Dn Dw/Dn Measured Dn Dw/Dn
particles (nm) particles (nm)
35 min 4637 7.3 2.63 103 63.6 1.6
Final 823 12.1 1.6 193 57.7 1.4
 Molecules 2014, 19 9

1
2
3
4

5 Figure 5. Micrograph and histograms of a sample taken at the end of the polymerization at
6 pH 1.75.

7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25 The results obtained in the second polymerization conducted at pH 1.75 allowed to know that two
26 particle populations are formed since the beginning of the polymerization and that the aggregation of a
27 fraction of small particles would occur along the polymerization. This suggests that probably the
28 monomer dosing rate is so low that the system is forced to generate very small but unstable particles.
29 To test this hypothesis a polymerization with a faster ECA dosing rate (0.017 g/min) was conducted,
30 since in accordance with the findings in semicontinuous heterophase polymerization at monomer
31 starved conditions [19-21] larger and more stable particles should be obtained, avoiding this way the
32 particle aggregation.
33 The micrograph and the corresponding histogram in Figure 6 show that indeed only one particle
34 population is observed when the polymerization was carried out at a faster monomer dosing rate. From
35 the measurement of more than 986 particles a Dn value of 42.3 nm and a polydispersity index of 1.4
36 were determined. Using the same calculation procedure previously employed, it was found that in this
37 case one molecule of surfactant covers approximately 1.6 nm2 of particle surface area, which is much
38 smaller value than those obtained in the polymerizations carried out at the slower monomer dosing
39 rate, indicating a higher particle stability. Only one population of larger and more stable particles
40 obtained as result of an increase in the monomer dosing rate confirm the hypothesis given above.
 Molecules 2014, 19 10

1
2
3
4
5

6 Figure 6. Micrograph and histograms of a sample taken at the end of the polymerization at
7 pH 1.75 and 0.017 g/min in monomer dosing rate.

8
9
10
11
12
13
14
15
16
17
18
19
20
21

22 3. Experimental Section

23 3.1. Reagents
24 ECA (99 %) was purchased from Sigma-Aldrich (Toluca, México) and stored at 4 °C; TW80 was
25 obtained from Oxiteno (Guadalajara, México); both of them were used as received. De-ionized and
26 triple-distilled water was drawn from a millipore system.
27
28 3.2. Polymerizations
29 The reactions were conducted in a 25 mL jacketed glass reactor equipped with a reflux condenser
30 and mechanical agitation following the procedure described below. 20 g water and 0.35 g TW80 were
31 charged into the reactor, after which the mixture was subjected to 450 rpm agitation and the
32 temperature stabilized in 35 °C; the reaction was started by the beginning of the monomer addition. In
33 all the polymerizations 2 g ECA were added using two different time periods, that is to say, 4 and 2
34 hours, which equals to 0.008 and 0.016 g/min. Monomer addition was done manually by dripping
35 measured amounts of ECA over the micellar solution each 5 minutes. After the end of the monomer
36 addition the reaction was allowed to proceed for a further period of 3 hours. The water pH in the
37 polymerizations was initially adjusted to 1.0, 1.5 and 1.75 for the ECA addition period of 4 h, while
38 for the period of 2 h, only the adjustment to pH 1.75 was carried out. The monomer conversion at the
39 time of sampling was determined by gravimetry.
 Molecules 2014, 19 11

1
2 3.3. Characterization
3 Particle size. The determinations of particle the size distributions for the samples of the latexes were
4 carried out in a JEOL JSM-7401F scanning-transmission electron microscope (STEM). For the
5 measurements a dilution containing about 2.5 g polymer per liter was prepared, depositing one drop of
6 it on a copper grid and allowing to dry. The diameters of a variable number of particles were measured
7 from the set of micrographs using an image analysis program (ImageJ 1.37c). From these data, Dw, Dn
8 and PDI (Dw/Dn), being Dw and Dn the weight- and number-average diameters and PDI the
9 polydispersity index, were calculated using the following equations:
10
11
12

13 Dn =
∑ nD
i i i
=
∑n D i i
(2)
∑ n i i n
14
4

15 Dw =
∑ i n i Di
(3)
3
∑ i ni Di
16

17 4. Conclusions

18 Semicontinuous heterophase polymerization at monomer starved conditions conducted at different

19 initial pH´s in the range 1 to 1.75 allowed to obtain latexes containing very small particles of PECA
20 and solid contents around 10.5 %, which are substantially higher than those reported in the literature
21 on preparing PACA latexes. Irrespective of the polymerization pH, two particle populations are
22 formed since the early stages of the polymerization, with the smaller Dn values at the end of the
23 reaction resulting from that carried out at pH 1.75, in this case, 11.9 and 58.9 nm, for the small and
24 large particles populations, respectively. The micrographs indicate that larger particles are formed by
25 the aggregation of a fraction of the smaller ones, which preserve their individuality. A polymerization
26 conducted at a pH 1.75 and faster monomer dosing rate than that used in the rest of the reactions
27 allowed to obtain particles with a Dn value near to 40 nm, distributed in only one population. This
28 finding indicates that while it is possible to obtain smaller particles when the monomer is dosed at a
29 very low rate, they are instable, thereby a fraction of them aggregates to form a population of larger
30 particles, increasing in this way the latex stability. Taken into account the potential use of
31 biocompatible and biodegradable particles with diameters smaller than 50 nm, the results reported
32 herein could be used as a basis to investigate on pH´s and monomer dosing rate using TW80 or more
33 effective surfactants that allow to prepare latexes with stable particles smaller than 40 nm in average
34 diameter preserving the relatively high solids content attained in this study.

35 Aknowledgments
 Molecules 2014, 19 12

1 National Council of Science and Technology (CONACyT) supported this research through grants
2 2013-223227 and 232753 (Laboratorio Nacional de Materiales Grafénicos). One of the authors (C.
3 Barrera) aknowledges the scholarship from CONACyT. The authors are grateful to Marcelina Sánchez
4 for their technical assistance in characterization work.
5

6 Author Contributions

7 HS: design and writing of paper; ST and CB: experimentation and results discussion; JS and YG:
8 support in characterization and its results discussion; RGL: support in design and writing the paper.

9 Conflicts of Interest

10 The authors declare no conflict of interest

11

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1 Sample Availability: Samples of the compounds are not available from the authors.

2 © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
3 distributed under the terms and conditions of the Creative Commons Attribution license
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