16. - 18. 10. 2013, Brno, Czech Republic, EU
PREPARATION OF NANOPARTICLES BASED ON BIODEGRADABLE CHAIN LINKED
PLA/PEG POLYMER FOR CONTROLLED RELEASE OF HERBICIDES METAZACHLOR
Pavel KUCHARCZYK, Alena PAVELKOVÁ, Petr STLOUKAL, Marek KOUTNÝ,
Vladimír SEDLAŘÍK
1 Tomas
Bata University in Zlin, Zlin, Czech Republic, EU, [email protected]
Abstract
The work presented here investigates the synthesis of PLA-PEG diisocyanate chain-linked copolymer, and
its application in the nano-encapsulation of bioactive compounds. Nanoparticle formation was performed via
a single solvent evaporation process, and the particles obtained were characterized by dynamic light
scattering. Results show the low molecular weight nature of the material with the glass transition
temperatures at around 44 °C. Nanoparticles in the range of 300 nm contained metazachlor were
successfully prepared, and their releasing behaviour exhibited first order release kinetics. The
biodegradability of material was proved by degradation under compost conditions.
Keywords: polylactide copolymer, nanoparticles, encapsulation, metazachlor
1.
INTRODUCTION
In the last several decades, bioactive compound delivery systems have attracted increasing interest in
various fields like biomedical, environmental and agriculture. In agriculture, such controlled release
formulations, including particles loaded with various agrochemicals, could prevent the unwanted phenomena
associated with conventional applications of agrochemicals such as leaching through the soil, volatility, and
degradation. Simultaneously, they could extend their activity in soil, improve their stability, and reduce
unwanted toxicity [1, 2].
Especially polylactic acid (PLA) and its copolymers are suitable for these types of applications. The utilization
of PLA as a matrix in microparticle release systems has been widely investigated through various techniques
of encapsulation including emulsification−diffusion method, nanoprecipitation, supercritical antisolvent
coprecipitation and so on. Among these, the oil in water solvent evaporation technique proved to be the most
preferred encapsulation method due to its relative simplicity, with no need for specialized equipment [3].
It is well known that entrapping efficiency and releasing pattern of bioactive agent from polymer matrix is a
function of various parameters especially type of polymer – drug and their interactions, polymer molecular
weight and experimental conditions (pH, temperature) [4].
In this work we examined the utilization of newly prepared PLA-polyethylene glycol (PEG) chain linked
copolymer (PLA-PEG-CL) for encapsulation and releasing of herbicide metazachlor (MTZ). As the chain
linker, aromatic diizocyanate 4,4′-Methylenebis(phenyl isocyanate) (MDI) was used. Based on our
preliminary work we found out that this type of material exhibits higher hydrophobicity than neat PLA and
therefore might be potentially more suitable for entrapping of hydrophobic MTZ. The polymer was prepared
by melt polycondensation of PLA in presence of PEG followed by chain linking with MDI. For encapsulation
simple oil in water solvent evaporation technique was applied. For description of encapsulation process
dynamic light scattering measurements and high performance liquid chromatography were used.
2.
MATERIAL AND METHODS
L-lactic acid (LA), 80% water solution and PEG, (Mw= 380 – 420 g∙mol-1) were sourced from Merck,
Hohensbrunn, Germany. Tin(II) 2-ethylhexanoate (Sn(Oct)2), ~95; 4,4′-methylenebis(phenyl isocyanate),
16. - 18. 10. 2013, Brno, Czech Republic, EU
(MDI), 98%, were purchased from Sigma Aldrich, Steinheim, Germany. Solvents - chloroform, acetone,
methanol and ethanol (all analytical-grade) - were obtained from IPL Petr Lukes, Uhersky Brod, the Czech
Republic. Chloroform (HPLC-grade) was bought from Chromspec, Brno, the Czech Republic. Metazachlore
grade was the same as in our previous work [5].
2.1
Synthesis of PLA-PEG-CL polymer
100 mL of L-LA was added into a 250 mL two-neck distillation flask equipped with a Teflon stirrer. The flask
was then connected to a condenser and placed in an oil bath. Firstly, dehydration of L-LA solution at 160°C
took place, under the reduced pressure of 20 kPa for 4 hours. Then, 0.5 wt.% Sn(Oct) 2 and 7.5 wt.% PEG
were added and the reaction continued for 6 hours at 10 kPa. After that, the pressure was reduced to 3 kPa
for another 10 hours. The resultant hot melt was poured out on aluminium foil and cooled; this pre-product
was labelled as prepolymer.
30 g of this product was added into a 250 mL two-neck flask equipped with a mechanical stirrer. The material
was slowly heated to the pre-determined temperature of 160 °C, under an N2 atmosphere. Once the mixture
had completely melted MDI was added (5.56 g) and the reaction was conducted for 30 minutes. The
resultant product was cooled, dissolved in acetone, precipitated into a water/methanol mixture (1:1), then
filtered and dried in a vacuum at 30 °C for 24 hours.
2.2
Analytical methods
For determination of molecular weight and thermal properties, gel permeation chromatography (GPC) and
differential scanning calorimetry (DSC) were conducted. The experimental conditions for both experiments
were the same as described in [6].
2.3
Preparation of nanoparticles
The method for this was adopted from a previous study by the authors, with only a few modifications being
effected [5]. The dry precipitated product (0.1 g) and 20 % of MTZ (calculated by polymer weight) were
dissolved in 1 mL of chloroform and emulsified in water containing PVA (0.5 %). The mixture was dispersed
under continuous stirring at 18 000 rpm for 5 min (homogenizer DI 18 basic, Yellow Line by IKA, Belgium).
Then, the emulsion was sonicated by an ultrasonic probe (Hielscher UP 400S, Germany) for 5 min with the
amplitude set to 35 %. Finally, the organic solvent was evaporated under reduced pressure (20 kPa) with
stirring, and the suspension of nanoparticles was formed. In order to determine the average diameter of
particles the dynamic light scattering method (Zetasizer Nano, Malvern instruments, Worcestershire, UK)
was utilized.
Encapsulation efficiency (EE, in %) and herbicide loading (HL, in %) were calculated as described in
previous work by the authors [5].
2.4
Releasing experiment
5 mL of re-suspended particle suspensions were transferred into 100 mL of phosphate buffer (20 mmol∙L −1,
pH=7) containing 0.2 % of sodium azide to prevent undesirable microbial degradation (Figure 2-E). All this
was carried out in triplicate. Suspensions were shaken (120 rpm) at 25 °C. Subsamples of 1.5 mL were
taken at time intervals (0-720 h), centrifuged at 14 000 rpm for 10 min, and filtered through a 0.22 μm syringe
PTFE filter to remove any remaining particles. The MTZ in samples was determined by the HPLC method
described in reference [5].
2.5
Biodegradation in compost
This experiment was conducted according to conditions described elsewhere [7]. Just very briefly, the
polymer sample was mixed with compost medium in gas tight flask. Evolved CO2 was measured by gas
16. - 18. 10. 2013, Brno, Czech Republic, EU
chromatography in predetermined time intervals. The temperature was set to during the whole experiment
56 °C.
3.
RESULTS AND DISCUSSION
Molecular and thermal characteristics of synthesized polymer are summarized in Table 1. It can be seen that
molecular weight was increased after chain linking reaction with MDI from initial 5 200 to final 39 300 g∙mol-1.
It shows on successful reaction between pre-polymer end groups with isocyanate. Thermal analysis revealed
that final product did not exhibited melting behaviour which suggests its amorphous nature due to presence
of aromatic rings in the structure. The glass transition temperature was detected above 44 °C which was
above the temperature of releasing experiment and therefore the effect of increased chain mobility could be
neglected.
Table 1 GPC and DSC characteristics of synthesized products.
Mw [g∙mol-1]
DM
Tga [°C] Cp [J∙g-1∙K-1]
pre-polymer
5200
1.6
15.3
0.504
PLA-PEG-CL
39300
2.2
44.3
0.485
a – data taken from second heating scan
b – data taken from first heating scan
n.d – not detected
Tmb [°C]
101.1
n.d
ΔHm [J∙g-1]
-32.3
n.d
The results from nanoparticles preparation and MTZ encapsulation are shown in Table 2. As can be noticed,
the used oil in water solvent evaporation technique is suitable for obtaining the particles in nanometer scale.
Polydisperzity index (PDI) was also relatively narrow which is desirable for predicting of behaviour of the
whole system.
Table 2 Characteristics of the nanoparticles obtained and encapsulated MTZ.
diameter [nm] s.d. [nm]
PDI*
s.d.
EE [%]
s.d. [%]
PLA-PEG-CL
320
4
0.18
0.015
64.7
1.4
* - polydisperzity based on DLS measurement
HL [%]
11.5
s.d. [%]
0.3
Encapsulation efficiency described the amount of MTZ entrapped inside the particles was nearly 65 % and
herbicide loading 11.5 %. Since the theoretical HL should be 20 % it can be concluded, that the rest amount
of MTZ remained in the solution during encapsulation. This is interesting to compare the results from
encapsulation experiment with the results determined in the previous work by the authors, where only neat
PLA was used. Under the same conditions of encapsulation its affectivity was 59 % which was lower than in
case of PLA-PEG-CL copolymer. It should be also notice that the diameter of particles prepared in this work
was lower than in case of neat PLA (~600 nm). The lower particle diameter along with higher EE show on
better ability of PLA-PEG-CL copolymer to form nanoparticles and better affinity of MTZ to this type of matrix
rather than to neat PLA.
MTZ releasing profile is depicted in Figure 1. It can be seen that immediately after the particles were put into
liquid buffered medium some MTZ was released (t=0). This was attributed to MTZ entrapped too close to the
particle surface. Releasing profiles displayed first order kinetics, and it could be seen that a relatively high
portion of MTZ still remained in the nanoparticles after completing the experiment. This was probably
trapped inside the nanoparticles, therefore, diffusion out was restricted. Comparison of results with these
obtained with neat PLA polymer and presented in [5] there was one significant difference. The initial release
in this work (10 %) was considerably lower than in case of neat PLA (35 %), at the same experimental
conditions. This also shows on better affinity of MTZ to this type of polymer.
16. - 18. 10. 2013, Brno, Czech Republic, EU
Figure 1 – Release profile of MTZ from PLA-PEG-CL nanoparticles.
The results from biodegradation experiment depicted in Figure 2 should prove the biodegradability of
material. In can be seen, that prepared PLA-PEG-CL exhibited biodegradability and the rate of mineralization
was considerably slower than cellulose (internal reference). It can be also seen, that mineralization at the
end of the experiment reached value around 50 % which was considerable low than it is typical for neat PLA
(around 70 % in 50 days) [8].
Fig. 2 Biodegradation of PLA-PEG-CL polymer in compost.
16. - 18. 10. 2013, Brno, Czech Republic, EU
4.
CONCLUSIONS
New copolymer based on PLA containing PEG units was successfully prepared by chain linking of low
molecular weight PLA-PEG with diisocyanate compound as the chain linker. Material was used for
encapsulation of herbicide metazachlor. Oil in water solvent evaporation technique used for encapsulation
provided nanorpaticles in dimensions of 300 nm with MTZ encapsulation efficiency 65 %. The releasing
experiment show that initial fast release of MTZ was reduced in contrast with neat PLA and releasing pattern
exhibited 1st order kinetic. The prepared material show certain level of biodegradability under composting
conditions.
ACKNOWLEDGEMENTS
This work has been supported by Operational Programme Research and Development for
Innovations, co-funded by the European Regional Development Fund (project
CZ.1.05/2.1.00/03.0111). The authors are also grateful to the Internal Grant Agency of Tomas Bata
University in Zlin (grant IGA/FT/2013/004) for co-funding. This work was also supported by the Grant
Agency of Czech Republic (Grant GACR P108/10/0200)
LITERATURE
[1]
QUAGLIA, F., BARBATO, F., DE ROSA, G., GRANATA, E., MIRO, A. LA, ROTONDA, M. I. Reduction of the
environmental impact of pesticides: waxy microspheres encapsulating the insecticide carbaryl. Journal of
Agricultural and Food Chemistry, 2001, vol. 49, p. 4808−4012.
[2]
SOPENA, F., CABRERA, A., MAQUEDA, C., MORÁLKO, E. Controlled release of the herbicide norflurazon into
water from ethylcellulose formulations. Journal of Agricultural and Food Chemistry, 2005, vol. 53, p. 3540−3547.
[3]
ROUZES, C., LEONARD, M., DURAND, A., DELLACHERIE, E. Influence of stabilizing agents and preparative
variables on the formation of poly(D,L-lactic acid) nanoparticles by an emulsification-diffusion technique.
Colloids Surfaces ,B 2003, vol. 32, p. 125−135.
[4]
ZHAO, J.; WILKINS, R. M. Low molecular weight polylactic acid as a matrix for the delayed release of
pesticides. Journal of Agricultural and Food Chemistry, 2005, vol. 53, p. 4076−4082.
[5]
STLOUKAL, P., KUCHARCZYK, P., SEDLARIK, V., BAZANT, P., KOUTNY, M. Low Molecular Weight
Poly(lactic acid) Microparticles for Controlled Release of the Herbicide Metazachlor: Preparation, Morphology,
and Release Kinetics. Journal of agricultural and food chemistry, 2012, vol. 60, p. 4111-4119.
[6]
KUCHARCYZK, P., POLJANSEK, I., SEDLARIK, V. The Effect of Various Catalytic Systems on Solid-State
Polymerization of Poly-(L-lactic acid). Journal of Macromolecular Science, Part A: Pure and Applied Chemistry,
2012, vol. 49., p.795-805.
[7]
STLOUKAL, P., VERNEY, V., COMMEREUC, S., RYCHLY, J., MATISOVA-RYCHLA, L., PIS, V., KOUTNY, M.
Assessment of the interrelation between photooxidation and biodegradation of selected polyesters after artificial
weathering. Chemosphere, 2012, vol. 88, p.1214-1219.
[8]
SUPRAKAS, S., R., MOSTO, B. Biodegradable polymers and their layered silicate nanocomposites: In greening
the 21st century materials world. Progress in Materials Science, 2005, vol. 8, p. 962-1079.
Download

PREPARATION OF NANOPARTICLES BASED ON