Research Article Preparation and Loading with Rifampicin...

Research ArticlePreparation and Loading with Rifampicin of Sub-50 nmPoly(ethyl cyanoacrylate) Nanoparticles by SemicontinuousHeterophase Polymerization

H Saade1 C Barrera1 R Guerrero2 E Mendizaacutebal3 J E Puig4 and R G Loacutepez1

1Departamento de Procesos de Polimerizacion Centro de Investigacion en Quımica Aplicada Boulevard Enrique Reyna No 14025294 Saltillo COAH Mexico2Sıntesis de Polımeros Centro de Investigacion en Quımica Aplicada Boulevard Enrique Reyna No 14025294 Saltillo COAH Mexico3Departamento de Quımica Universidad de Guadalajara BoulevardM Garcıa-Barragan No 1451 44430 Guadalajara JAL Mexico4Ingenierıa Quımica Universidad de Guadalajara Boulevard M Garcıa-Barragan No 1451 44430 Guadalajara JAL Mexico

Correspondence should be addressed to R G Lopez guillermolopezciqaedumx

Received 2 May 2016 Revised 23 June 2016 Accepted 28 June 2016

Academic Editor Md Nurunnabi

Copyright copy 2016 H Saade et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We report the preparation of poly(ethyl cyanoacrylate) (PECA) nanoparticles by semicontinuous heterophase polymerizationcarried out at monomer starved conditions at three monomer addition rates Particles in the nanometer range were obtainedthe size of which diminishes with decreasing monomer addition rate as shown by the fact that particles with mean diameters ofca 42 and 30 nm were obtained at the faster and intermediate dosing rates respectively whereas two populations of particles oneof 155 and the other of 36 nm in mean diameters were produced at the slower dosing rate The obtained molecular weights werefrom 2200 to 3500 gmol depending on the addition rate which are typical of the anionic polymerizations of cyanoacrylates inaqueous dispersions at low pHs The rifampicin (RIF) loading into the nanoparticles was successful since the entire drug addedwas incorporated The drug release study carried out at pH of 72 indicated a faster release from the free RIF at intermediate andlarger release times as expected since in the nanoparticles first the drug has to diffuse through the nanoparticle structure Thecomparison of several drug release models indicates that the RIF release from PECA nanoparticles follows that of Higuchi

1 Introduction

Poly(alkyl cyanoacrylates) PACA are a family of biodegrad-able polymers that are being intensively investigated forthe development of drug delivery nanoparticles [1ndash3] Thesepolymeric nanostructures are usually obtained by anionicemulsion polymerization [2] Couvreur et al first reportedthe emulsion polymerization of the alkyl cyanoacrylates inparticular of methyl cyanoacrylate and ethyl cyanoacrylate(ECA) obtaining latexes that contained nanoparticles withmean diameter of 200 nm and less than 1 in solid content[4] These authors also reported the nanoparticles loadingwith fluorescein and daunorubicin being the first to proposePACA nanoparticles as drug carriers In the following yearsthe research on PACA nanoparticles was significant [5ndash14]

rendering fundamental knowledge about the conditions andmechanisms involved in their preparation In parallel anumber of reports on drug-loaded PACA nanoparticles alsoappeared [3 15ndash19]

The reports on PACA nanoparticles preparation haveallowed knowing that the type and concentration of surfac-tant [5ndash10] and the initial pH [5 9 11ndash14] mainly determinethe particle size Different types of Dextrans [5 9 11ndash13] and Tweens (TW) [4 5 10 11] are the most testedsurfactants the latter being the ones that enable obtainingthe smaller PACA nanoparticle sizes On this particularissue it could be appointed that Behan et al [12] obtainedpoly(butyl cyanoacrylate) (PBCA) nanoparticles with meandiameter around 400 nm using Dextran 70 at pHs between2 and 3 On the other hand Douglas et al [5] prepared

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2016 Article ID 8384973 11 pageshttpdxdoiorg10115520168384973

2 Journal of Nanomaterials

PBCA nanoparticles with average particle diameter close to50 nm by polymerizing in emulsions stabilized with Tween60 (TW60) at pH of 225 Larger particles were obtainedwhen Tweens (TW20 and TW40) with lower molecularweights were employed which suggests an inverse relation-ship between particle size and Tween molecular weight Thework of Yang et al [8] is remarkable because of the verysmall PBCA particles reported The authors show micro-graphs where particles with diameters smaller than 50 nmare identified however they mention that there is anotherpopulation composed of much larger particles (not shown inthe micrographs) Unfortunately the authors do not includeparticle size histograms to know more precisely the sizes ofthe obtained particles Incidentally the absence of particlesize distributions from electronmicroscopymeasurements inthe quotedworks is noticeable [4ndash14] being themean particlesizes reported those obtained by quasi-elastic light scattering(QLS)

Recently our group documented the synthesis ofpoly(ethyl cyanoacrylate) PECA nanoparticles by a tech-nique named as semicontinuous heterophase polymerizationat monomer-starved conditions [20] Here we demonstratedthat modulating the monomerdosing rate it is possibleto obtain latexes containing only one population of verysmall particles In fact a latex composed of PECA particleswith 423 nm in mean diameter determined by electronmicroscopy and a solid content close to 10 was obtainedSuch small particle size together with relatively highsolid content is not common in the synthesis of PACAnanoparticles In fact mean particle diameters over 100 nmare usual in reports on drug-loaded PACA nanoparticles[2 3 15ndash19] Nevertheless nanoparticles ranging from 10to 50 nm in diameter are very attractive as drug deliverycarriers because of the expected increase in their efficacy[21 22] Their smallness would increase the ability to crossthrough intestinal walls entering the blood stream [21] andreducing their clearance by the immunological system [23]

In continuation of our previous work herein we reportthe preparation of PECA nanoparticles with mean diametersmaller than 40 nmachieved bymodulating the dosing rate ofmonomer Furthermore the same surfactant previously usedTween 80 (TW80) as well as the pH at which the smallerparticle size was obtained 175 was used in the present workLoading of PECA nanoparticles with rifampicin (RIF) andthe corresponding release study were also included RIF afirst-line antituberculosis drug was chosen because of thegreat interest in the development of more efficacy ways fordrug administration as well as to reduce the dose frequency[24]

2 Materials and Methods

21 Materials ECA (99) dichloromethane (995) andspectrophotometric grade chloroform were purchased fromSigma-Aldrich (Mexico) HPLC-grade tetrahydrofuran(THF) was provided from Merck TW80 was obtained fromOxiteno (Guadalajara Mexico) RIF was obtained fromLupin Tarapur (India) All chemicals were used as received

Deionized and triple-distilled water was drawn from a T SBarnstead E-Pure 4-Holder Water Purification System

22 Polymerizations Reactions were conducted in a 25mLjacketed glass reactor equipped with a reflux condenserand mechanical agitation following the procedure describedbelow 20 g of water adjusted to pH of 175 using a mixtureof phosphates and 035 g of TW80 were charged into thereactor after which the mixture was subjected to 450 rpmagitation and the temperature stabilized at 35∘C the reactionstarted with the beginning of the monomer addition In allpolymerizations 2 g of ECA was added at different dosingrates (119865

119900) (00055 0011 and 0017 gmin) with a dosing pump

(Kd Scientific-100) The polymerizations were stopped at theend of the dosing period by turning off thewater recirculationsystem used for maintaining the reaction temperature andthen transferring the latex to a vessel previously cooledin an ice-water bath Due to the relatively large sampleamounts required for the characterizations a series of runswere carried out for each polymerization For a given 119865

119900

the addition period in the polymerization was divided into5 intervals then a run for each interval was carried outstopping the polymerization and collecting all the latex at theend of each of them to determine conversion by gravimetryand molecular weight Particle size was only measured in thefinal latex For the dosing rate of 0005 gmin the intervals 12 3 4 and 5 correspond to values of the normalized dosingtime 119905

119903 equal to 020 040 062 083 and 1 respectively here

the normalized time is the ratio of the sampling time 119905dividedby the total addition time For the dosing rate of 0017 gminthe 119905119903values for the same intervals were 021 042 063 083

and 1 With this protocol the amount of latex obtained wasenough to make all the required characterizations Samplesduring the polymerization at 0011 gminwere not taken onlythe final latex was analyzed

23 Characterization231 Particle Size Determination of the particle size distri-butions for latexes samples was carried out in a JEOL JSM-7401F field emission scanning electronmicroscope (FE-SEM)operated at scanning-transmission mode (STEM) using atransmission electron detector SM74230RTD For the mea-surements a dilution containing about 25 g of solids per literwas prepared depositing one drop of it on a copper grid andallowed to dry After that the sampleswere stainedwith a dropof a 2wt aqueous solution of phosphotungstic acid Thediameters of a variable number of particles were manuallymeasured one by one from the set of micrographs withthe image analysis program ImageJ 137c From these dataparticle size histograms were elaborated and 119863

119908 119863119899 and

PDI (= 119863119908119863119899) 119863119908and 119863

119899being the weight- and number-

average diameters respectively and PDI the polydispersityindex were calculated using the following equations

119863119899=sum119894

119899119894119863119894

sum119894

119863119894

=sum119899119894119863119894

119899

119863119908=sum119894

1198991198941198634119894

sum119894

1198991198941198633119894

(1)

Journal of Nanomaterials 3

232 Molecular Weight Determination For measuring themolecular weight distribution (MWD) and the averagemolecular weights of the polymer the latexes samples werelyophilized and dissolved in HPLC-grade THF used asthe mobile phase An Agilent PL-GPC50 gel permeationchromatograph (GPC) equippedwith three columns (AgilentPLgel) and a refractive index detector was employed TheGPC was calibrated with polystyrene (PS) standards (Poly-science) covering the molecular weight range of 60 times 102to 30 times 106 gmol The reported average molecular weightswere those provided by the equipment so they are relative topolystyrene

24 Chemical Structure Analysis

Nuclear Magnetic Resonance (NMR) Spectroscopy The poly-mer samples were analyzed by 1H (16 scans) and 13C (12000scans) NMR in a Bruker-400MHz spectrometer Acetone-d6was used as solvent and the analyses were performed at roomtemperature

Fourier Transform Infrared (FTIR) Spectroscopy This anal-ysis was carried out in a Spectrophotometer ThermoNicoletMagna-550 For this a sample of the dried polymericmaterial was grinded with potassium bromide powder andsubsequently pressed to form a disk which was analyzed inthe apparatus

25 RIF Loading Process The procedure to load the drugwithin the nanoparticles was as follows 575 g of a latexfrom a polymerization carried out at 0011 gmin was dilutedwith 1025 g of water and then charged again into the reactorwhere the polymerization was performed After that theaddition of a 06 g of RIF-DCM solution with 15 wt drugwas initiatedmaintaining the dispersion at 25∘Cand 450 rpmagitation The addition was carried out by intermittent shotsof the solution allowing the dispersion to recover the originalappearance before the next shot The total addition timewas 260min The latex obtained from the loading processwas filtered through a 02120583m filter and a sample of thefiltered latex was taken for RIF concentration determinationsThe RIF concentration in the nanoparticles was determinedwith a Shimadzu multiespec-1501UV-vis spectrophotometerby analyzing the dried solid resulting from the lyophilizedsample dissolved in spectrophotometric grade chloroformFor this purpose a calibration curve was elaborated rangingfrom 10 to 100 ppm of RIF by dissolving the required quanti-ties of the drug in spectrophotometric grade chloroform andreading the absorbance at 348 nmThe analysis of the samplerequired the dissolution of 2mg of loaded nanoparticles in10mL of spectrophotometric grade chloroform

26 In Vitro RIF Release Studies These studies were car-ried out using the dialysis technique For this procedure26mg of RIF-loaded nanoparticles dispersed in 2mL ofphosphate buffer solution at pH of 72 was loaded intoa dialysis membrane bag (12500 gmol in exclusion size)which was then sealed After that the prepared membrane

XX

i(

)

0

20

40

60

80

100

02 04 06 08 1000tr

Figure 1 Evolution of instantaneous 119883119894

(mdash) and global 119883 (- - -)conversions with relative time for the polymerizations of ECA at twomonomer dosing rates 00055 (X) and 0017 (◼) gmin

was introduced into a 05 L phosphate buffer solution at pH of72 applying intense magnetic agitation and maintaining thesystem temperature at 37∘C One mL samples of the buffersolution were then taken at predetermined times duringthe test and analyzed from the intensity of the absorbanceband at 334 nm in a Shimadzu multiespec-1501UV-vis spec-trophotometer After each sampling 1mL of the buffer wasadded to the buffer solution to maintain the original volumeFor comparison a sealed dialysis membrane bag containing10mg of RIF dispersed in 2mL of phosphate buffer solutionat pH of 72 was also immersed into 05 L of the same buffersolution to determine the profile of the drug dissolution inthe buffer under similar conditions to that used with theRIF-loaded nanoparticles and the same sampling sequenceThe release tests were carried out in duplicate for RIF-loadednanoparticles and in triplicate for free-RIF

It is worth mentioning that RIF instability in humanplasma (pH72 to 74) is well knownHowever it is also knownthat RIF maintains its antibacterial activity between pH val-ues from 55 to 8 [25] as demonstrated by the number of yearsthat this drug has been used systematically against tubercu-losis Literature is plenty of reports on RIF-nanostructurespreparation which includes drug release studies In thesestudies RIF instability is not taken into account as it does notaffect the release profiles Furthermore the main method todetermine RIF concentration in the releasing medium is UVspectrophotometry because the selected signal to follow suchconcentration is not affected by the change in the chemicalstructure of drug [26ndash28]

3 Results and Discussion

31 Kinetics Figure 1 shows the instantaneous conversion(119883119894) and the global conversion (119883) as a function of the

normalized time (119905119903) for the runs made at the slowest

(00055 gmin) and fastest (0017 gmin) addition rates The


(a)

0

50

100

150

200

250

300

Num

ber f

requ

ency

10 20 30 40 50 60 700D (nm)

(b)

Figure 2 Representative STEM micrograph (a) and the corresponding particle size histogram (b) of a sample of PECA latex from thepolymerization at a monomer dosing rate of 0011 gmin

instantaneous conversion is the fraction of added monomerup to time 119905 that has changed into polymer the global con-version is defined as the fraction of total monomer convertedto polymer at time 119905 and 119905

119903is the ratio of the sampling

time 119905 divided by the total addition time as indicated in theExperimental Section this normalization allows comparingthe curves in Figure 1 at the same overall fraction of addedmonomer inasmuch as the effective addition times differsubstantiallyThis plot clearly reveals that119883

119894increases rapidly

with reaction time reaching values near 80 for the slowestaddition rate and 100 for the slowest one when 119905

119903asymp

02 High instantaneous conversions indicate that monomer-starved conditions were achieved for bothmonomer additionrates while the difference between the 119883

119894curves up to 119905

119903asymp

06 suggests that there is an inverse relationship betweenmonomer dosing rate and instantaneous conversion Atthe end of the polymerization however the instantaneousconversion at the slowest dosing rate falls below the oneattained for the fastest dosing rate which as we will showand discuss below is related to the agglomeration of thesmall particles to produce another population of largerparticles Our group reported the above mentioned inverserelationship for the free-radical semicontinuous heterophasepolymerization (SHP) of several monomers [29ndash31] but toour knowledge our previous paper [20] and this one arethe first reports on anionic SHP of alkyl cyanoacrylates inaqueous dispersions stabilized with surfactants As we haveexplained elsewhere [29ndash31] in monomer-starved condi-tions the volumetric growth of the particles diminishes asthe dosing rate is reduced leading as a consequence to theformation of a larger number of particles of smaller size andfaster depletion of fed monomer [32]

32 Particle Size Table 1 depicts the final 119863119899and PDI

values obtained from the runs made at the different dosing

Table 1 Average particle size and particle size polydispersity fromthe latexes obtained at the end of the polymerizations of ECA carriedout at different monomer dosing rates

Dosing rate(gmin)

Population 1 Population 2119863119899

(nm) PDI 119863119899

(nm) PDI00170 423 14 mdash mdash00110a 273 13 mdash mdash00110b 304 12 mdash mdash00055 155 11 362 11Original run (a) and its replicate (b)

rates employed Similarly to results obtained from otherSHP reports [29ndash31] particle size decreases as the dosingrate diminishes from 00170 to 00055 gmin Elsewhere wereported the data for the fastest dosing rate (0017 gmin)which revealed that only one particle population with423 nm in 119863

119899was obtained [20] Here the particle sizes we

obtained from the two polymerizations at the dosing rate of0011 gmin were similar since the measured mean diameterswere 273 and 304 nm at final conversion for the original runand for the replica respectively However and in contrast tothe results at 0017 gmin the size distribution obtained at theslower dosing rate indicates a bimodal size population with119863119899= 155 nm for one population and 362 nm for the other

one Previously we have suggested that the larger particleswere produced by the aggregation of a fraction of the smallerones in order to preserve the stability of the dispersion alongthe polymerization [20]

Figures 2 and 3 depict micrographs and the correspond-ing histograms of the polymerizationsmade at dosing rates of0011 and 00055 gmin respectively The micrograph shownin Figure 2 reveals the presence of spheroidal particles withcertain degree of dispersion as indicated by the PDI of 13 and


(a)

0102030405060708090

100110120130140150

Num

ber f

requ

ency

2 4 6 8 10 12 14 16 18 20 22 24 260D (nm)

(b)

0102030405060708090

100110120130140150

Num

ber f

requ

ency

5 10 15 20 25 30 35 40 45 50 55 60 65 70 750D (nm)

(c)

Figure 3 Representative STEMmicrograph (a) of a sample of PECA latex from the polymerization at amonomer dosing rate of 00055 gminHistograms of the small (b) and large (c) particle populations identified are included

12 for the two polymerizations (the original and its replicate)estimated from the data in the histograms The micrographin Figure 3 shows a more polydispersed particle distributionwhere two populations can be observed and revealed by thehistograms obtained from a set of micrographs Howeverthe low polydispersity of each one of the populations isnoticeable

Previously we have reported that for the dosing rate of0017 gmin the particle surface covered by one molecule ofTW80was 16 nm2 and that amonomodal particle populationof 42 nm in 119863

119899was produced hence we concluded that the

surface coverage ratio was sufficiently small to stabilize theformed particles [20] The procedure to estimate this surfacecoverage ratio consisted of first calculating the numberconcentration of particle (119873

119901) in the latex with the following

equation

119873119901=6119862119901

1205871205881199011198633119899

(2)

where 119862119901is the polymer concentration in the latex (gmL of

water) and 120588119901the PECA density (11 gmL) [20] The number

of surfactant molecules per mL of water (119873119904) was calculated

from the molecular weight of the surfactant (1310 gmol)and its weight used in the formulation employed for thepolymerization The values of 119873

119901and 119873

119904calculated in this

way for the latex made at the dosing rate of 0011 gminallowed estimating that 28 and 21 nm2 of particle surfacefor the original run and its replicate respectively werecovered by one surfactant molecule Although these val-ues are higher than that calculated for the dosing rate of0017 gmin (16 nm2) they are still small enough to stabilizethe particles giving only one population For the slowestdosing rate two-particle size populations were produced onewith average number particle size of 155 nm and the otherone with 362 nm The surface coverage area per molecule ofTW80 was calculated from the average particle size of thesmaller particle population giving a value of 36 nm2 whichappears to be too large to produce a single population ofparticles These results suggest that it is possible to reduce


0

500

1000

1500

2000

2500

3000

3500

MnM

w(g

mol

)

20 40 60 80 1000X ()

(a)

0

500

1000

1500

2000

2500

3000

3500

MnM

w(g

mol

)

20 40 60 80 1000X ()

(b)

Figure 4 Evolution of average molecular weights119872119908

(◼) and119872119899

(◻) in the polymerizations of ECA at monomer dosing rates of 0017 (a)and 00055 (b) gmin

the particle size through a decrease in the monomer dosingrate however as the rate decreases the surface coverage areaincreases Under the recipe and conditions used in this studyvalues as high as 28 nm2 per surfactant molecule still allowobtaining a single particle population However when thisratio increased to 36 nm2 two particle populations wereformed This indicates that between 28 and 36 nm2 thereis a threshold limit value for the particle surface covered byonemolecule of TW80 above which a fraction of the particlemust aggregate to form another population and to increasethe system stability

From the direct relationship between particle size andmonomer addition rate we can elucidate the kinetic behaviorof the polymerizations At the slowest addition rate a largernumber density of smaller particles is produced yielding alarger number of reaction sites and as a consequence fasterreaction rates which is evident at 02 le 119905

119903le 06 However

for 119905119903gt 06 119883

119894diminishes due to the aggregation that

produces another population of larger particles bringing as aresult a reduction of the reaction sites For the fastest additionrate the increase of 119883

119894at 119905119903gt 06 could be related to the

increment of the number density of particles which shouldincrease the number of reaction sites Moreover unlike thepolymerization at slowest addition rate there is no evidencehere of particle aggregation Literature reports of SHP ofacrylic monomers have demonstrated that the formation ofparticles exists even at the final stages of polymerization [29ndash31] which is in agreement with results presented here

33 Molecular Weight It has been reported that molecularweights of PACA synthesized in dispersed aqueous mediumare quite small El-Egakey et al [33] studied the effect of

surfactant concentration on themolecular weight of this kindof polymers and found weight-average molecular weights(119872119908) between 1400 and 3800 gmol Seijo et al [6] polymer-

ized isobutyl and isohexyl cyanoacrylates and obtained valuesof 119872119908of ca 2500ndash3000 gmol 119872

119908obtained here are also

on the order of 2500 gmol throughout the reaction for theaddition rate of 0017 gmin with119872

119908119872119899values119872

119899being

the number-averagemolecular weight of ca 11 (Figure 4(a))for the slowest addition rate 119872

119908is ca 3250 gmol up to

conversions ca 40 and then it drops to values around2750 gmol at larger conversions (Figure 4(b)) A similarbehaviour is observed in the case of 119872

119899for the slowest

addition rate Furthermore a low value (11) for 119872119908119872119899is

also notedIn the emulsion polymerization of alkyl cyanoacrylates it

is recognized that the growth of the polymeric chains endswhen they enter in contact with a H+ from the aqueousmedium [12] However it is not clear whether the protonfrom the aqueous medium penetrates into the particle or theactive extreme of the propagation chain and the proton meetnear the surface of the growing particleThe larger molecularweights obtained at the slowest addition rate in the earlierstages of the reaction could have its origin in the large numberdensity of small particles produced in this period on thecontrary the low molecular weights at the fastest additionrate might be due to the smaller number density of particlesThis should bring as a result a larger ratio of particles toprotons in the aqueous medium at the slowest addition ratecompared to the fastest addition one which should resultin a decrease in the frequency at which the protons interactwith the active polymeric particles yielding consequently adecrease in the frequency of termination and largermolecularweights However the aggregation of particles at the slowest


C

C

NC

O

(ppm)

O

a

b

c

c

a

b

Acetone-d6

65

65

60

60

55

55

50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

05

05

00

00

70

70

CH2

CH3

CH2

(a)

C

NC

OC

O

d

h

i

e

fg

i

eg

h

160

160

150

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140

140

130

130

120

120

110

110

100

100 9090 8080 7070 6060 5050 4040 3030 2020 1010 00

170

170

Acetone-d6

d + f

CH2

CH3

CH2

(ppm)

(b)

Figure 5 1H (a) and 13C (b) NMR spectra of a sample of the polymer from the polymerization at a monomer dosing rate of 0011 gmin

addition rate and conversions larger than 40 reduces thenumber density of particles and consequently the ratio ofreactive particles to protons causing a drop in molecularweight

As pointed out previously in the polymerization ofalkyl cyanoacrylates in aqueous dispersions stabilized withsurfactants it is usual to obtain polymers with lowmolecularweights This could be due to a high termination rate ofgrowing chains as a consequence of the large availability ofH+ when the reaction is done at low pHs The work of Daset al [7] favors this hypothesis these authors polymerizedisobutyl cyanoacrylate in aqueous dispersions stabilized witha surfactant and at pHs between 2 and 7 the obtainedvalues of119872

119899were between 1000 and 13000 gmol when the

reactions were conducted at pH of 2 but at pH of 7 theobtained119872

119899values were between 26000 and 600000 gmol

Das et al argued that at low pH values due to the highconcentration of H+ the growing period of the chains is veryshort and as a consequence oligomers or polymers of lowermolecular weight are formed whereas at higher pHrsquos theconcentration of H+ is lower which leads to a longer polymerchain growing and larger molecular weights

34 Chemical Structure Analysis Figure 5 displays the 1Hand 13C NMR spectra of the polymeric material obtainedat the end of the original polymerization carried out at0011 gminThe similarity of these spectra with that reportedfor PECA in literature is noteworthy [34] The 1H NMRspectrum shows the absence of the peaks of the olefinicprotons at 120575 65 and 120575 75 ppm indicating that there is nounpolymerized monomer The broad peaks at 120575 26ndash30 ppm(a) are ascribed to the methylene group in the backbone ofthe chain while the peaks at 120575 43 (b) and 120575 13 ppm (c) aredue to the protons of pending methylene and methyl groupsrespectively Meanwhile the signals detected in the 13CNMRspectra were 120575 116 (e) assigned to the carbon in the cyanogroup the peaks at120575 43 to120575 47 ppmwhich corresponds to thecarbon of methylene and quaternary carbon in the backbonechain (d + f) the peak at 120575 167 ppm (g) due to the carbon

0

20

40

60

80

100

Tran

smitt

ance

()

3500 3000 2500 2000 1500 1000 5004000Wavenumber (cmminus1

)

1254 cmminus11750 cmminus1

2248 cmminus1

2990 cmminus1

Figure 6 FTIR spectrum of a sample of the polymer from thepolymerization at a monomer dosing rate of 0011 gmin

in the carboxyl group and the peaks at 120575 65 ppm (h) and 12057514 ppm (i) related to the carbons of the pendant methyleneand methyl groups respectively

The FTIR spectra corresponding to the polymeric mate-rial collected at the end of the original polymerization carriedout at 0011 gmin are shown in Figure 6 This spectra matchwell with those reported for PECA in the literature [34 35]In this spectra the peak at 2990 cmminus1 which is ascribed tothe symmetric and asymmetric C-H stretching vibrations ofmethylene and methyl groups in the pendant ethyl the peakat 2248 cmminus1 due to the CequivN stretching at 1750 cmminus1 a peakcorresponding to the C=O group stretching and finally thepeak at 1254 cmminus1 attributed to the C-O stretching can beidentified The absence of absorbance at around 3130 cmminus1signal ascribed to C=CH2 stretching in the monomer [35] isevident

In this way it can be concluded that the detected signals inthe NMR and FTIR spectrum in Figures 5 and 6 respectively


20 40 60 80 1000RIF concentration (ppm)

0

05

1

15

2

25

3

Abso

rban

ce

Figure 7 UV calibration curve for RIF dissolved in spectrophotometric grade chloroform at an absorbance of 348 nm Fit equation 119910 =00275119909 minus 00055 1198772 = 09999

demonstrate that PECA was obtained in the polymerizationscarried out in this study

35 RIF Loading in Particles and Release Tests For the drugloading of the particles we used the latex obtained fromthe polymerization carried out at 0011 gmin which reached976 conversion with 11 of total solids According to theemployed recipe the expected content of RIF if the entiredrug was loaded would be 126 For its part the sample ofRIF-loaded particles analyzed by UV-vis spectrophotometrygave a mean absorbance of 06817 at 348 nm in wavelengthwhich corresponds to 125 of drug in the particles accord-ing to the calibration curve (Figure 7) This indicates that theentire drug charged was loaded in the PECA particles withinthe experimental error

RIF-loaded particles were not characterized by STEMtaken into account that there is no reason to change theparticle size distribution as a result of the RIF loadingA simple calculation assuming additive volumes indicatesthat the increase in the particle diameter resulting from theincorporation of RIF to reach 125 wt in the particles isabout only 46 This is equivalent to an increase of about13 nm in119863

119899 moreover there is no reason to expect a change

in the PDI of PECA nanoparticlesFigure 8 depicts the results of the drug release tests from

the free RIF dispersed in 2mL phosphate buffer solution inthe bag dialysis and from theRIF-loaded particlesThe releaseprofiles are similar for both systems at short release times (lt 4hours) but then they depart the drug release being higher inthe free RIF as expected since the drug into the nanoparticleshas to diffuse through the polymer chains of the nanoparticlesto reach the wall of the dialysis membrane After 10 hoursthe drug released from the bag containing free RIF is evenlarger than the one released from the nanoparticles in factafter 26 hours most of the free RIF have been released

0

20

40

60

80

100

RIF

rele

ased

()

5 10 15 20 25 30 350t (h)

Figure 8 Drug release profiles from RIF-loaded PECA nanoparti-cles (◼) and free RIF (998771)

whereas only 70 of the drug has been released from thenanoparticles after 30 hours Elsewhere the relatively rapiddrug release from nanoparticles compared to larger particleshas been documented [36] We propose here to explain thisfaster release that the distance that the drug must diffuse toreach the surface is shorter especially for the molecules thatare closer to the surface moreover this release time couldbe reduced if the drug forms a solid solution in the particlesinstead of aggregating to form crystals

To determine the mechanism of drug release from theparticles several models were compared in particular thoseof order zero and order one Higuchi and Korsmeyer-Peppas[37] The drug release data was plotted in the linearizedform of each of these models (Figure 9) According to theplots shown in this figure and taking into account the meanstandard deviation of the data used to make the linearizedform of these models the Higuchi model gave the best fittingcorrelation of the data The equation of the Higuchi model isthe following [37]

119862 = 11987011990512

(3)


0

20

40

60

80

100RI

F re

leas

ed (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(a)

0002040608101214161820

log

RIF

rem

aini

ng (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(b)

0102030405060708090

100

RIF

rele

ased

()

5 10 15 20 25 30 35 40 450t12 (min12)

(c)

0002040608101214161820

log

RIF

rele

ased

()

15 20 25 30 35 4010log t (min)

(d)

Figure 9 Release models tested for RIF released from PECA nanoparticles zero order (a) first order (b) Higuchi (c) and Korsmeyer-Peppas(d) Fit equations 119910 = 0034119909 + 1453 1198772 = 0896 (a) 119910 = minus0000277119909 + 19425 1198772 = 0959 (b) 119910 = 1706119909 minus 0516 1198772 = 0979 (c) and119910 = 0525119909 + 0177 1198772 = 0948 (d)

Here 119862 is the drug release 119870 the kinetic constant and119905 the measured release time Even though we expected thatthe best model to reproduce the drug release was that ofKorsmeyer-Peppas because it considers both drug diffusionand particle erosion the drug release follows a Fick diffusionmechanism in accordance with Higuchirsquos model due only toa concentration gradient within the system [38] As is wellknown the particle erosion comes from the hydrolysis of thependant ester groups of PECA which given the obtainedresults did not take place during the period of the releasetests [39] However it is also known that PECA hydrolysisis promoted mainly by enzymes such as those in the bloodplasma which were absent in the release studies [39] Thisfact could explain why the Fick diffusionmodel fits better theexperimental data reported here

4 Conclusions

A method to prepare PECA nanoparticles with mean diam-eter around 30 nm dispersed in a latex containing about11 of total solids was disclosed To our best knowledgethere are no previous reports in the specialized literatureshowing the preparation of latexes containing so small PACAparticles in conjunctionwith a solid content as high as the oneobtained in this workThe study reveals that by decreasing themonomer addition rate it is possible to obtain even smallerparticles however at the slowest addition rate employedhere a fraction of them aggregate to finally give a bimodalpopulation of sizes The PECA nanoparticles were loadedwith RIF obtaining a loading efficiency of 100 and 126of drug content The release tests showed that RIF diffusesslower from the nanoparticles in comparison with that from

a dialysis bag containing the free drug following a Fickdiffusion mechanism in accordance with the Higuchi model

Competing Interests

The authors do not report conflict of interests whatsoever

Acknowledgments

National Council of Science and Technology (CONACyT)supported this research through Grants 2014-223227 and232753 (Laboratorio Nacional de Materiales Grafenicos) CBarrera acknowledges the scholarship from CONACyT Theauthors are grateful to Juan U Pena Jose L de la Pena andJudith Cabello for their technical assistance

References

[1] A Graf A McDowell and T Rades ldquoPoly(alkycyanoacrylate)nanoparticles for enhanced delivery of therapeuticsmdashis therereal potentialrdquo Expert Opinion on Drug Delivery vol 6 no 4pp 371ndash387 2009

[2] G Yordanov ldquoPoly (alkil cyanoacrylate) nanoparticles as drugcarriers 33 years laterrdquo Bulgarian Journal of Chemistry vol 1no 2 pp 61ndash72 2012

[3] C Vauthier D Labarre and G Ponchel ldquoDesign aspects ofpoly(alkylcyanoacrylate) nanoparticles for drug deliveryrdquo Jour-nal of Drug Targeting vol 15 no 10 pp 641ndash663 2007

[4] P Couvreur B M Kante M Roland P Guiot P Bauduin andP Speiser ldquoPolycyanoacrylate nanocapsules as potential lyso-somotropic carriers preparation morphological and sorptivepropertiesrdquo Journal of Pharmacy and Pharmacology vol 31 no1 pp 331ndash332 1979


[5] S J Douglas L Illum and S S Davis ldquoParticle size and sizedistribution of poly(butyl 2-cyanoacrylate) nanoparticles IIInfluence of stabilizersrdquo Journal of Colloid and Interface Sciencevol 103 no 1 pp 154ndash163 1985

[6] B Seijo E Fattal L Roblot-Treupel and P Couvreur ldquoDesign ofnanoparticles of less than 50 nm diameter preparation charac-terization and drug loadingrdquo International Journal of Pharma-ceutics vol 62 no 1 pp 1ndash7 1990

[7] S K Das I G Tucker D J T Hill and N Ganguly ldquoEvaluationof poly(isobutylcyanoacrylate) nanoparticles for mucoadhesiveocular drug delivery I Effect of formulation variables onphysicochemical characteristics of nanoparticlesrdquo Pharmaceu-tical Research vol 12 no 4 pp 534ndash540 1995

[8] S C Yang H X Ge Y Hu X Q Jiang and C Z Yang ldquoForma-tion of positively charged poly(butyl cyanoacrylate) nanoparti-cles stabilized with chitosanrdquo Colloid and Polymer Science vol278 no 4 pp 285ndash292 2000

[9] I Bertholon-Rajot D Labarre and C Vauthier ldquoInfluenceof the initiator system ceriumndashpolysaccharide on the surfaceproperties of poly(isobutylcyanoacrylate) nanoparticlesrdquo Poly-mer vol 46 no 4 pp 1407ndash1415 2005

[10] C K Weiss U Ziener and K A Landfester ldquoA route to non-functionalized and functionalized poly(n-butylcyanoacrylate)nanoparticles preparation in miniemulsionrdquo Macromoleculesvol 40 no 4 pp 928ndash938 2007

[11] S J Douglas L Illum S S Davis and J Krueter ldquoParticle sizeand size distribution of poly(butyl-2-cyanoacrylate) nanoparti-cles I Influence of physicochemical factorsrdquo Journal of Colloidand Interface Science vol 101 no 1 pp 149ndash158 1984

[12] N Behan C Birkinshaw and N Clarke ldquoPoly n-butyl cyano-acrylate nanoparticles a mechanistic study of polymerisationand particle formationrdquo Biomaterials vol 22 no 11 pp 1335ndash1344 2001

[13] C Chauvierre D Labarre and P Couvreur ldquoRadical emulsionpolymerization of alkylcyanoacrylates initiated by the redoxsystem dextran-cerium (IV) under acidic aqueous conditionsrdquoMacromolecules vol 36 no 16 pp 6018ndash6027 2003

[14] A Bootz T Russ F Gores M Karas and J Kreuter ldquoMolecularweights of poly(butyl cyanoacrylate) nanoparticles determinedby mass spectrometry and size exclusion chromatographyrdquoEuropean Journal of Pharmaceutics and Biopharmaceutics vol60 no 3 pp 391ndash399 2005

[15] J Wu and J-H Duan ldquoStudy of co-encapsulated doxorubicinand curcumin poly(butyl cyanoacrylate) nanoparticles andreversion of multidrug resistance in MCF-7ADR cell linerdquoJournal of Chemical and Pharmaceutical Research vol 5 no 10pp 415ndash423 2013

[16] S Snipstad S Westroslashm Y Moslashrch M Afadzi A Aslund andC de Lange Davies ldquoContact-mediated intracellular deliveryof hydrophobic drugs from polymeric nanoparticlesrdquo CancerNanotechnology vol 5 no 1 article 8 2014

[17] H E Shahmabadi F Movahedi M K M Esfahani et al ldquoEffi-cacy of Cisplatin-loaded polybutyl cyanoacrylate nanoparticleson the glioblastomardquo Tumor Biology vol 35 no 5 pp 4799ndash4806 2014

[18] S K B Doun S E Alavi S K B Esfahani H E ShahmabadiF Alavi and S Hamzei ldquoEfficacy of Cisplatin-loaded poly butylcyanoacrylate nanoparticles on the ovarian cancer an in vitrostudyrdquo Tumor Biology vol 35 no 8 pp 7491ndash7497 2014

[19] X Zhao W Li Q Luo and X Zhang ldquoEnhanced bioavail-ability of orally administered flurbiprofen by combined use

of hydroxypropyl-cyclodextrin and poly(alkyl-cyanoacrylate)nanoparticlesrdquo European Journal of Drug Metabolism and Phar-macokinetics vol 39 no 1 pp 61ndash67 2014

[20] H Saade S Torres C Barrera J Sanchez Y Garza and R GLopez ldquoEffect of pH and monomer dosing rate in the anionicpolymerization of ethyl cyanoacrylate in semicontinuous oper-ationrdquo International Journal of Polymer Science vol 2015 ArticleID 827059 9 pages 2015

[21] A Des Rieux V Fievez M Garinot Y-J Schneider and VPreat ldquoNanoparticles as potential oral delivery systems of pro-teins and vaccines a mechanistic approachrdquo Journal of Con-trolled Release vol 116 no 1 pp 1ndash27 2006

[22] M Elsabahy and K L Wooley ldquoDesign of polymeric nanopar-ticles for biomedical delivery applicationsrdquo Chemical SocietyReviews vol 41 no 7 pp 2545ndash2561 2012

[23] D Thassu M Deleers and Y Pathak Nanoparticulate DrugDelivery System InformaHealthcare NewYork NY USA 2007

[24] L L I J Booysen L Kalombo E Brooks et al ldquoIn vivoin vitropharmacokinetic and pharmacodynamic study of spray-driedpoly-(dl-lactic-co-glycolic) acid nanoparticles encapsulatingrifampicin and isoniazidrdquo International Journal of Pharmaceu-tics vol 444 no 1-2 pp 10ndash17 2013

[25] httpswwwsigmaaldrichcomcontentdamsigma-aldrichdocsSigmaProduct Information Sheet1r3501pispdf

[26] L Balaita V Maier L Verestiuc and M Popa ldquoPolymer nano-particles for release of rifampicinrdquo Advanced Science Engineer-ing and Medicine vol 5 no 2 pp 96ndash104 2013

[27] M Rajan and V Raj ldquoFormation and characterization ofchitosan-polylacticacid-polyethylene glycol-gelatin nanoparti-cles a novel biosystem for controlled drug deliveryrdquo Carbohy-drate Polymers vol 98 no 1 pp 951ndash958 2013

[28] D Pooja L Tunki H Kulhari B B Reddy and R Sistla ldquoChar-acterization biorecognitive activity and stability of WGAgrafted lipid nanostructures for the controlled delivery ofRifampicinrdquo Chemistry and Physics of Lipids vol 193 pp 11ndash172015

[29] R LedezmaM E Trevino L E Elizalde et al ldquoSemicontinuousheterophase polymerization under monomer starved condi-tions to prepare nanoparticles with narrow size distributionrdquoJournal of Polymer Science Part A Polymer Chemistry vol 45no 8 pp 1463ndash1473 2007

[30] M G Perez-Garca M Rabelero S M Nuno-Donlucas etal ldquoSemicontinuous heterophase polymerization of n-butylmethacrylate effect ofmonomer feeding raterdquo Journal ofMacro-molecular Science Part A Pure and Applied Chemistry vol 49no 7 pp 539ndash546 2012

[31] M G Perez-Garcıa A G Alvarado M Rabelero et al ldquoSem-icontinuous heterophase polymerization of methyl and hexylmethacrylates to produce latexes with high nanoparticles con-tentrdquo Journal of Macromolecular Science Part A Pure andApplied Chemistry vol 51 no 2 pp 144ndash155 2014

[32] J J Krackeler and H Naidus ldquoParticle size and molecularweight distributions of various polystyrene emulsionsrdquo Journalof Polymer Science Part C vol 27 no 1 pp 207ndash235 1969

[33] M A El-Egakey V Bentele and J Kreuter ldquoMolecular weightsof polycyanoacrylate nanoparticlesrdquo International Journal ofPharmaceutics vol 13 no 3 pp 349ndash352 1983

[34] M G Han S Kim and S X Liu ldquoSynthesis and degradationbehavior of poly(ethyl cyanoacrylate)rdquo Polymer Degradationand Stability vol 93 no 7 pp 1243ndash1251 2008


[35] G Yordanov and Z Bedzhova ldquoPoly(ethyl cyanoacrylate) col-loidal particles tagged with Rhodamine 6G preparation andphysicochemical characterizationrdquo Central European Journal ofChemistry vol 9 no 6 pp 1062ndash1070 2011

[36] D P Otto and M M de Villiers ldquoWhy is the nanoscale special(or not) Fundamental properties and how it relates to thedesign of nano-enabled drug delivery systemsrdquoNanotechnologyReviews vol 2 no 2 pp 171ndash199 2013

[37] MH Shoaib J Tazeen H AMerchant and R I Yousuf ldquoEval-uation of drug release kinetics from ibuprofen matrix tabletsusing HPMCrdquo Pakistan Journal of Pharmaceutical Sciences vol19 no 2 pp 119ndash124 2006

[38] C Vauthier C Dubernet E Fattal H Pinto-Alphandary andP Couvreur ldquoPoly(alkylcyanoacrylates) as biodegradable mate-rials for biomedical applicationsrdquo Advanced Drug DeliveryReviews vol 55 no 4 pp 519ndash548 2003

[39] P Couvreur and C Vauthier ldquoPolyalkylcyanoacrylate nanopar-ticles as drug carrier present state and perspectivesrdquo Journal ofControlled Release vol 17 no 2 pp 187ndash198 1991

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


PBCA nanoparticles with average particle diameter close to50 nm by polymerizing in emulsions stabilized with Tween60 (TW60) at pH of 225 Larger particles were obtainedwhen Tweens (TW20 and TW40) with lower molecularweights were employed which suggests an inverse relation-ship between particle size and Tween molecular weight Thework of Yang et al [8] is remarkable because of the verysmall PBCA particles reported The authors show micro-graphs where particles with diameters smaller than 50 nmare identified however they mention that there is anotherpopulation composed of much larger particles (not shown inthe micrographs) Unfortunately the authors do not includeparticle size histograms to know more precisely the sizes ofthe obtained particles Incidentally the absence of particlesize distributions from electronmicroscopymeasurements inthe quotedworks is noticeable [4ndash14] being themean particlesizes reported those obtained by quasi-elastic light scattering(QLS)

Recently our group documented the synthesis ofpoly(ethyl cyanoacrylate) PECA nanoparticles by a tech-nique named as semicontinuous heterophase polymerizationat monomer-starved conditions [20] Here we demonstratedthat modulating the monomerdosing rate it is possibleto obtain latexes containing only one population of verysmall particles In fact a latex composed of PECA particleswith 423 nm in mean diameter determined by electronmicroscopy and a solid content close to 10 was obtainedSuch small particle size together with relatively highsolid content is not common in the synthesis of PACAnanoparticles In fact mean particle diameters over 100 nmare usual in reports on drug-loaded PACA nanoparticles[2 3 15ndash19] Nevertheless nanoparticles ranging from 10to 50 nm in diameter are very attractive as drug deliverycarriers because of the expected increase in their efficacy[21 22] Their smallness would increase the ability to crossthrough intestinal walls entering the blood stream [21] andreducing their clearance by the immunological system [23]

In continuation of our previous work herein we reportthe preparation of PECA nanoparticles with mean diametersmaller than 40 nmachieved bymodulating the dosing rate ofmonomer Furthermore the same surfactant previously usedTween 80 (TW80) as well as the pH at which the smallerparticle size was obtained 175 was used in the present workLoading of PECA nanoparticles with rifampicin (RIF) andthe corresponding release study were also included RIF afirst-line antituberculosis drug was chosen because of thegreat interest in the development of more efficacy ways fordrug administration as well as to reduce the dose frequency[24]

2 Materials and Methods

21 Materials ECA (99) dichloromethane (995) andspectrophotometric grade chloroform were purchased fromSigma-Aldrich (Mexico) HPLC-grade tetrahydrofuran(THF) was provided from Merck TW80 was obtained fromOxiteno (Guadalajara Mexico) RIF was obtained fromLupin Tarapur (India) All chemicals were used as received

Deionized and triple-distilled water was drawn from a T SBarnstead E-Pure 4-Holder Water Purification System

22 Polymerizations Reactions were conducted in a 25mLjacketed glass reactor equipped with a reflux condenserand mechanical agitation following the procedure describedbelow 20 g of water adjusted to pH of 175 using a mixtureof phosphates and 035 g of TW80 were charged into thereactor after which the mixture was subjected to 450 rpmagitation and the temperature stabilized at 35∘C the reactionstarted with the beginning of the monomer addition In allpolymerizations 2 g of ECA was added at different dosingrates (119865

119900) (00055 0011 and 0017 gmin) with a dosing pump

(Kd Scientific-100) The polymerizations were stopped at theend of the dosing period by turning off thewater recirculationsystem used for maintaining the reaction temperature andthen transferring the latex to a vessel previously cooledin an ice-water bath Due to the relatively large sampleamounts required for the characterizations a series of runswere carried out for each polymerization For a given 119865

119900

the addition period in the polymerization was divided into5 intervals then a run for each interval was carried outstopping the polymerization and collecting all the latex at theend of each of them to determine conversion by gravimetryand molecular weight Particle size was only measured in thefinal latex For the dosing rate of 0005 gmin the intervals 12 3 4 and 5 correspond to values of the normalized dosingtime 119905

119903 equal to 020 040 062 083 and 1 respectively here

the normalized time is the ratio of the sampling time 119905dividedby the total addition time For the dosing rate of 0017 gminthe 119905119903values for the same intervals were 021 042 063 083

and 1 With this protocol the amount of latex obtained wasenough to make all the required characterizations Samplesduring the polymerization at 0011 gminwere not taken onlythe final latex was analyzed

23 Characterization231 Particle Size Determination of the particle size distri-butions for latexes samples was carried out in a JEOL JSM-7401F field emission scanning electronmicroscope (FE-SEM)operated at scanning-transmission mode (STEM) using atransmission electron detector SM74230RTD For the mea-surements a dilution containing about 25 g of solids per literwas prepared depositing one drop of it on a copper grid andallowed to dry After that the sampleswere stainedwith a dropof a 2wt aqueous solution of phosphotungstic acid Thediameters of a variable number of particles were manuallymeasured one by one from the set of micrographs withthe image analysis program ImageJ 137c From these dataparticle size histograms were elaborated and 119863

119908 119863119899 and

PDI (= 119863119908119863119899) 119863119908and 119863

119899being the weight- and number-

average diameters respectively and PDI the polydispersityindex were calculated using the following equations

119863119899=sum119894

119899119894119863119894

sum119894

119863119894

=sum119899119894119863119894

119899

119863119908=sum119894

1198991198941198634119894

sum119894

1198991198941198633119894

(1)








XX

i(

)

0

20

40

60

80

100

02 04 06 08 1000tr










(a)

0

50

100

150

200

250

300

Num

ber f

requ

ency

10 20 30 40 50 60 700D (nm)

(b)







119903asymp



119903asymp





Dosing rate(gmin)


(nm) PDI 119863119899








(a)

0102030405060708090

100110120130140150

Num

ber f

requ

ency

2 4 6 8 10 12 14 16 18 20 22 24 260D (nm)

(b)

0102030405060708090

100110120130140150

Num

ber f

requ

ency

5 10 15 20 25 30 35 40 45 50 55 60 65 70 750D (nm)

(c)







equation

119873119901=6119862119901

1205871205881199011198633119899

(2)





119901and 119873




0

500

1000

1500

2000

2500

3000

3500

MnM

w(g

mol

)

20 40 60 80 1000X ()

(a)

0

500

1000

1500

2000

2500

3000

3500

MnM

w(g

mol

)

20 40 60 80 1000X ()

(b)


(◼) and119872119899




119903le 06 However

for 119905119903gt 06 119883










119908119872119899values119872

119899being









C

C

NC

O

(ppm)

O

a

b

c

c

a

b

Acetone-d6

65

65

60

60

55

55

50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

05

05

00

00

70

70

CH2

CH3

CH2

(a)

C

NC

OC

O

d

h

i

e

fg

i

eg

h

160

160

150

150

140

140

130

130

120

120

110

110

100

100 9090 8080 7070 6060 5050 4040 3030 2020 1010 00

170

170

Acetone-d6

d + f

CH2

CH3

CH2

(ppm)

(b)









0

20

40

60

80

100

Tran

smitt

ance

()


)


2248 cmminus1

2990 cmminus1







0

05

1

15

2

25

3

Abso

rban

ce








0

20

40

60

80

100

RIF

rele

ased

()

5 10 15 20 25 30 350t (h)




119862 = 11987011990512

(3)


0

20

40

60

80

100RI

F re

leas

ed (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(a)

0002040608101214161820

log

RIF

rem

aini

ng (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(b)

0102030405060708090

100

RIF

rele

ased

()

5 10 15 20 25 30 35 40 450t12 (min12)

(c)

0002040608101214161820

log

RIF

rele

ased

()

15 20 25 30 35 4010log t (min)

(d)



4 Conclusions



Competing Interests


Acknowledgments


References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










XX

i(

)

0

20

40

60

80

100

02 04 06 08 1000tr










(a)

0

50

100

150

200

250

300

Num

ber f

requ

ency

10 20 30 40 50 60 700D (nm)

(b)







119903asymp



119903asymp





Dosing rate(gmin)


(nm) PDI 119863119899








(a)

0102030405060708090

100110120130140150

Num

ber f

requ

ency

2 4 6 8 10 12 14 16 18 20 22 24 260D (nm)

(b)

0102030405060708090

100110120130140150

Num

ber f

requ

ency

5 10 15 20 25 30 35 40 45 50 55 60 65 70 750D (nm)

(c)







equation

119873119901=6119862119901

1205871205881199011198633119899

(2)





119901and 119873




0

500

1000

1500

2000

2500

3000

3500

MnM

w(g

mol

)

20 40 60 80 1000X ()

(a)

0

500

1000

1500

2000

2500

3000

3500

MnM

w(g

mol

)

20 40 60 80 1000X ()

(b)


(◼) and119872119899




119903le 06 However

for 119905119903gt 06 119883










119908119872119899values119872

119899being









C

C

NC

O

(ppm)

O

a

b

c

c

a

b

Acetone-d6

65

65

60

60

55

55

50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

05

05

00

00

70

70

CH2

CH3

CH2

(a)

C

NC

OC

O

d

h

i

e

fg

i

eg

h

160

160

150

150

140

140

130

130

120

120

110

110

100

100 9090 8080 7070 6060 5050 4040 3030 2020 1010 00

170

170

Acetone-d6

d + f

CH2

CH3

CH2

(ppm)

(b)









0

20

40

60

80

100

Tran

smitt

ance

()


)


2248 cmminus1

2990 cmminus1







0

05

1

15

2

25

3

Abso

rban

ce








0

20

40

60

80

100

RIF

rele

ased

()

5 10 15 20 25 30 350t (h)




119862 = 11987011990512

(3)


0

20

40

60

80

100RI

F re

leas

ed (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(a)

0002040608101214161820

log

RIF

rem

aini

ng (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(b)

0102030405060708090

100

RIF

rele

ased

()

5 10 15 20 25 30 35 40 450t12 (min12)

(c)

0002040608101214161820

log

RIF

rele

ased

()

15 20 25 30 35 4010log t (min)

(d)



4 Conclusions



Competing Interests


Acknowledgments


References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a)

0

50

100

150

200

250

300

Num

ber f

requ

ency

10 20 30 40 50 60 700D (nm)

(b)







119903asymp



119903asymp





Dosing rate(gmin)


(nm) PDI 119863119899








(a)

0102030405060708090

100110120130140150

Num

ber f

requ

ency

2 4 6 8 10 12 14 16 18 20 22 24 260D (nm)

(b)

0102030405060708090

100110120130140150

Num

ber f

requ

ency

5 10 15 20 25 30 35 40 45 50 55 60 65 70 750D (nm)

(c)







equation

119873119901=6119862119901

1205871205881199011198633119899

(2)





119901and 119873




0

500

1000

1500

2000

2500

3000

3500

MnM

w(g

mol

)

20 40 60 80 1000X ()

(a)

0

500

1000

1500

2000

2500

3000

3500

MnM

w(g

mol

)

20 40 60 80 1000X ()

(b)


(◼) and119872119899




119903le 06 However

for 119905119903gt 06 119883










119908119872119899values119872

119899being









C

C

NC

O

(ppm)

O

a

b

c

c

a

b

Acetone-d6

65

65

60

60

55

55

50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

05

05

00

00

70

70

CH2

CH3

CH2

(a)

C

NC

OC

O

d

h

i

e

fg

i

eg

h

160

160

150

150

140

140

130

130

120

120

110

110

100

100 9090 8080 7070 6060 5050 4040 3030 2020 1010 00

170

170

Acetone-d6

d + f

CH2

CH3

CH2

(ppm)

(b)









0

20

40

60

80

100

Tran

smitt

ance

()


)


2248 cmminus1

2990 cmminus1







0

05

1

15

2

25

3

Abso

rban

ce








0

20

40

60

80

100

RIF

rele

ased

()

5 10 15 20 25 30 350t (h)




119862 = 11987011990512

(3)


0

20

40

60

80

100RI

F re

leas

ed (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(a)

0002040608101214161820

log

RIF

rem

aini

ng (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(b)

0102030405060708090

100

RIF

rele

ased

()

5 10 15 20 25 30 35 40 450t12 (min12)

(c)

0002040608101214161820

log

RIF

rele

ased

()

15 20 25 30 35 4010log t (min)

(d)



4 Conclusions



Competing Interests


Acknowledgments


References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a)

0102030405060708090

100110120130140150

Num

ber f

requ

ency

2 4 6 8 10 12 14 16 18 20 22 24 260D (nm)

(b)

0102030405060708090

100110120130140150

Num

ber f

requ

ency

5 10 15 20 25 30 35 40 45 50 55 60 65 70 750D (nm)

(c)







equation

119873119901=6119862119901

1205871205881199011198633119899

(2)





119901and 119873




0

500

1000

1500

2000

2500

3000

3500

MnM

w(g

mol

)

20 40 60 80 1000X ()

(a)

0

500

1000

1500

2000

2500

3000

3500

MnM

w(g

mol

)

20 40 60 80 1000X ()

(b)


(◼) and119872119899




119903le 06 However

for 119905119903gt 06 119883










119908119872119899values119872

119899being









C

C

NC

O

(ppm)

O

a

b

c

c

a

b

Acetone-d6

65

65

60

60

55

55

50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

05

05

00

00

70

70

CH2

CH3

CH2

(a)

C

NC

OC

O

d

h

i

e

fg

i

eg

h

160

160

150

150

140

140

130

130

120

120

110

110

100

100 9090 8080 7070 6060 5050 4040 3030 2020 1010 00

170

170

Acetone-d6

d + f

CH2

CH3

CH2

(ppm)

(b)









0

20

40

60

80

100

Tran

smitt

ance

()


)


2248 cmminus1

2990 cmminus1







0

05

1

15

2

25

3

Abso

rban

ce








0

20

40

60

80

100

RIF

rele

ased

()

5 10 15 20 25 30 350t (h)




119862 = 11987011990512

(3)


0

20

40

60

80

100RI

F re

leas

ed (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(a)

0002040608101214161820

log

RIF

rem

aini

ng (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(b)

0102030405060708090

100

RIF

rele

ased

()

5 10 15 20 25 30 35 40 450t12 (min12)

(c)

0002040608101214161820

log

RIF

rele

ased

()

15 20 25 30 35 4010log t (min)

(d)



4 Conclusions



Competing Interests


Acknowledgments


References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

500

1000

1500

2000

2500

3000

3500

MnM

w(g

mol

)

20 40 60 80 1000X ()

(a)

0

500

1000

1500

2000

2500

3000

3500

MnM

w(g

mol

)

20 40 60 80 1000X ()

(b)


(◼) and119872119899




119903le 06 However

for 119905119903gt 06 119883










119908119872119899values119872

119899being









C

C

NC

O

(ppm)

O

a

b

c

c

a

b

Acetone-d6

65

65

60

60

55

55

50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

05

05

00

00

70

70

CH2

CH3

CH2

(a)

C

NC

OC

O

d

h

i

e

fg

i

eg

h

160

160

150

150

140

140

130

130

120

120

110

110

100

100 9090 8080 7070 6060 5050 4040 3030 2020 1010 00

170

170

Acetone-d6

d + f

CH2

CH3

CH2

(ppm)

(b)









0

20

40

60

80

100

Tran

smitt

ance

()


)


2248 cmminus1

2990 cmminus1







0

05

1

15

2

25

3

Abso

rban

ce








0

20

40

60

80

100

RIF

rele

ased

()

5 10 15 20 25 30 350t (h)




119862 = 11987011990512

(3)


0

20

40

60

80

100RI

F re

leas

ed (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(a)

0002040608101214161820

log

RIF

rem

aini

ng (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(b)

0102030405060708090

100

RIF

rele

ased

()

5 10 15 20 25 30 35 40 450t12 (min12)

(c)

0002040608101214161820

log

RIF

rele

ased

()

15 20 25 30 35 4010log t (min)

(d)



4 Conclusions



Competing Interests


Acknowledgments


References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




C

C

NC

O

(ppm)

O

a

b

c

c

a

b

Acetone-d6

65

65

60

60

55

55

50

50

45

45

40

40

35

35

30

30

25

25

20

20

15

15

10

10

05

05

00

00

70

70

CH2

CH3

CH2

(a)

C

NC

OC

O

d

h

i

e

fg

i

eg

h

160

160

150

150

140

140

130

130

120

120

110

110

100

100 9090 8080 7070 6060 5050 4040 3030 2020 1010 00

170

170

Acetone-d6

d + f

CH2

CH3

CH2

(ppm)

(b)









0

20

40

60

80

100

Tran

smitt

ance

()


)


2248 cmminus1

2990 cmminus1







0

05

1

15

2

25

3

Abso

rban

ce








0

20

40

60

80

100

RIF

rele

ased

()

5 10 15 20 25 30 350t (h)




119862 = 11987011990512

(3)


0

20

40

60

80

100RI

F re

leas

ed (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(a)

0002040608101214161820

log

RIF

rem

aini

ng (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(b)

0102030405060708090

100

RIF

rele

ased

()

5 10 15 20 25 30 35 40 450t12 (min12)

(c)

0002040608101214161820

log

RIF

rele

ased

()

15 20 25 30 35 4010log t (min)

(d)



4 Conclusions



Competing Interests


Acknowledgments


References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





0

05

1

15

2

25

3

Abso

rban

ce








0

20

40

60

80

100

RIF

rele

ased

()

5 10 15 20 25 30 350t (h)




119862 = 11987011990512

(3)


0

20

40

60

80

100RI

F re

leas

ed (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(a)

0002040608101214161820

log

RIF

rem

aini

ng (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(b)

0102030405060708090

100

RIF

rele

ased

()

5 10 15 20 25 30 35 40 450t12 (min12)

(c)

0002040608101214161820

log

RIF

rele

ased

()

15 20 25 30 35 4010log t (min)

(d)



4 Conclusions



Competing Interests


Acknowledgments


References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

20

40

60

80

100RI

F re

leas

ed (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(a)

0002040608101214161820

log

RIF

rem

aini

ng (

)

250 500 750 1000 1250 1500 1750 20000t (min)

(b)

0102030405060708090

100

RIF

rele

ased

()

5 10 15 20 25 30 35 40 450t12 (min12)

(c)

0002040608101214161820

log

RIF

rele

ased

()

15 20 25 30 35 4010log t (min)

(d)



4 Conclusions



Competing Interests


Acknowledgments


References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials
















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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