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Summer Research Fellowship Programme of India's Science Academies

Optimization of Polymer Nanoparticle Synthesis and Characterisation by scattering techniques

V Vipul Kumar

Summer Research fellow, Bishopmoore college, Mavelikara 690101

Dr. V.K. Aswal

Head, MSAS section, SSPD, Bhabha Atomic Research centre, Mumbai 400085

ABSTRACT

Polymer nanoparticles obtained via nanoprecipitation methods are promising candidates for nanocarriers applications. The biomedical application of polymeric nanoparticles strongly depends on the particle size, which is decided by the different controlling factors that influence the underlining mechanism of particle formation. Specifically, nanoparticles of 150-300 nm are mainly going to the liver and the spleen while particles of 30-150 nm can be found in the heart, the kidney and, the stomach. Nanoparticles formed byPoly(lactic-co-glycolic acid) (PLGA), Polycaprolactone (PCL) are widely used due to their biodegradable properties. The change in the formation and size of PLGA and PCL nanoparticles in the presence of different factors e.g., solute concentration, solvent to non-solvent ratio, salt and urea have been examined by Dynamic Light Scattering (DLS) and Small Angle Neutron Scattering (SANS). Instead of relying on the hand mixing methods, nanoparticles are prepared using a controllable millifluidic setup. The final size of nanoparticles has been tuned by changing the solute concentration and solvent to non-solvent ratio in the nanoparticles. The presence of salt strongly influences the formation and final size of nanoparticles. A small amount of salt presence in the nanoparticles triggered the aggregation of nanoparticles. On the contrary, urea merely shows any effect in the size control of polymer nanoparticles.

Introduction

Polymeric Nanoparticles are particulate dispersion or solid particles with size ranging from 10-500 nm. Polymeric nanoparticles are researched widely as carriers in the pharmaceutical sector for controlled/sustained release in drug delivery systems. They offer a significant improvement over traditional oral and intravenous methods of administration in terms of efficiency and effectiveness.

Biodegradable polymers are a specific type of polymer that breaks down after its intended purpose to result in natural by products. These polymers are found both naturally and synthetically made, and largely consist of ester, amide, and ether functional groups. The biomedical application of polymeric nanoparticles strongly depends on the particle formation and their size [1]. Specifically, nanoparticles of 150-300 nm are mainly going to the liver and the spleen while particles of 30-150 nm can be found in the heart kidney and, stomach [2]. Therefore, in order to deliver the pharmaceutical agent to a desired location, the size and polydispersity of polymeric nanoparticles need to be controlled very precisely.

Delivers a higher concentration of pharmaceutical agent to a desired location, The choice of polymer and the ability to modify drug release from polymeric nanoparticles have made them ideal candidates for cancer therapy, delivery of vaccines, contraceptives and delivery of targeted antibiotics, Polymeric nanoparticles can be easily incorporated into other activities related to drug delivery, such as tissue engineering. Two main strategies used for preparation of polymeric nanoparticles are the “top-down” approach and the “bottom-up” approach. In the top-down approach a dispersion of preformed polymers produces polymeric nanoparticles, whereas in the bottom-up approach polymerization of monomers leads to the formation of polymeric nanoparticles. Various techniques can be used to produce polymer nanoparticles, such as solvent evaporation, salting-out, dialysis, supercritical fluid technology, micro-emulsion, mini-emulsion, surfactant-free emulsion, and interfacial polymerization. The choice of method depends on a number of factors, such as, particle size, particle size distribution, area of application, etc.

In the present work, the polymer nanoparticles are synthesizes by the nanoprecipitation or solvent displacement method. Nanoprecipitation is a simple, facile, mild and low energy input process to prepare the nanoparticles. Nanoprecipitation is rapid desolvation of the polymer the precipitation of nanoparticles occurs when the polymer solution is added to the non-solvent. The polymer-containing solvent diffuses into the dispersing medium, the polymer immediately precipitates. Various parameters like initial concentration, final concentration, mixing rate, solvent-nonsolvent ratio are varied during preparation of solutions. Because this method is a convenient, reproducible, fast and economic one step manufacturing for the preparation of polymeric Nanoparticles. Polycaprolactone (PCL) and Poly (lactic-co-glycolic acid) (PLGA) are synthesised and studied what parameters that affect the size of the polymer nanoparticles.

It is observed the size of nanoparticles is strongly associated with solvent to nonsolvent ratio. In addition, the amount of solute presence in the nanoparticles also plays an important role in the nanoparticles formation. Apart from that, the presence of salt significantly controls the size and stability of nanoparticles. The propensity of size and stability of nanoparticles changes with changing the functional group of initial polymers used for nanoparticles preparation. The presence of very small amount of NaCl (10 mM) destabilize Polycaprolacrone nanoparticles whereas nanoparticles obtained from Poly (lactic-co-glycolic acid) can be destabilized at higher NaCl (200 mM) concentration.

Literature Review

Many researchers put their attention in the field of synthesis and applications of polymeric nanoparticle synthesis. The nanoprecipitation followed by the formation of polymer nanoparticle synthesis involves some mechanisms.

Broichsitter et al 2010 reported that the Preparation of nanoparticles by solvent displacement method for drug delivery: A shift in the Ouzo region upon drug loading. The particle formation is based on the physicochemical principle called ouzo effect and Maragoni convection. This principle is useful to explain the particle formation [3].

G.J.L. Lebouille and Stepanyan reported the nanoprecipitation of polymers in a bad solvent. They studied Nanoprecipitation of polymers in a bad sol-vent in the presence of (polymeric) surfactants. The polymer is first dissolved in a good (organic) solvent, followed by a solvent switch towards a poor solvent environment in the presence of surfactant. The combined experimental and theoretical results on Nanoprecipitation demonstrate that diffusion limited coalescence is a mechanism that enables an adequate description of the nanoparticle formation process. In my work polymer is dissolved in the acetone to make a better interaction with the aqueous phase since acetone has high affinity towards water and its boiling point is low [4].

Philippe Legranda and Sylviane Lesieura reported Influence of polymer behaviour in organic solution on the production of polylactide nanoparticles by Nanoprecipitation [5]. The quality of the nanoparticles depends on the polymer–solvent interactions. Once a good solvent of the polymer has been chosen, structural properties of the polymer (chain length, chain ends, surface active properties determined by the polarity of the chain ends) are the main parameters which control size and fabrication yield. Thus, amphiphilic polymers are preferable because they remove the need to add surfactant to stabilize the interface. PCL and PLGA polymers are synthesised without using stabilizers.

Katsuji Noda et al 1980 reported Viscosities and densities at 298.15 K for mixtures of methanol, acetone, and water. In Nanoprecipitation organic phase consisting of polymer in acetone diffuses to water. Hence velocity of diffusion is affected by the viscosity of solution. Particle size is scaled by considering the viscosity of acetone water mixture at 25 ˚c from the data.

MATERIALS AND METHODS

Chemicals

Poly(lactic-co-glycolic acid),Polycaprolactone,Acetone, De-mineralised Water, Sodium Chloride, Urea

Structure of polymer used

Polycaprolactone(PCL)

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    PCL structure

    Poly (lactic-co-glycolic acid) (PLGA)

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      PLGA structure

      Method of Synthesis of Polymer Nanoparticles

      Nanoprecipitation method/ Solvent displacement method

      Nanoprecipitation is also called solvent displacement method. It involves the precipitation of a preformed polymer from an organic solution and the diffusion of the organic solvent in the aqueous medium in the presence or absence of a surfactant. The polymer is dissolved in a water-miscible solvent of intermediate polarity, leading to the precipitation of nanospheres. This phase is injected into a stirred aqueous solution. Polymer deposition on the interface between the water and the organic solvent, caused by fast diffusion of the solvent, leads to the instantaneous formation of a colloidal suspension.

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        Nanoprecipitation method

        PLGA and PCL polymer nanoparticles are synthesised using solvent displacement method (fig.1). PCL in Acetone (3mg/ml) is taken in a syringe having barrel capacity of 5 ml and a suitable volume is allowed to inject into a magnetically stirred (750 rpm) aqueous phase. The device used to inject the organic solution is known as millifluidic setup or Syringe pump injector (fig 2). The mode of the apparatus is set to be “Infuse only” and constant flow rate of 1mL/min. A particular target volume of polymer solution is injected to the aqueous phase. Nanoparticles of PCL gradually form after 10 minutes of stirring by diffusion of organic phase into aqueous phase.

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          Syringe pump 

          The process of particle formation in the Nanoprecipitation method includes three phases: nucleation, growth, and aggregation. Nanoparticles formation is a result surface tension gradient of two liquid phases. Liquid with a high surface tension (aqueous phase) pulls more strongly on the surrounding liquid than one with a low surface tension (organic phase).This difference between surface tensions of the aqueous and the oil phase causes interfacial turbulence and thermal inequalities in the system. This leads to the continuous formation of vortices of solvent at the interface of both liquids. The organic solvent diffuses from regions of low surface tension which causes gradual precipitation of the polymer nanoparticles.

          Dynamic Light Scattering (DLS)

          Theoretical Background of DLS

          Dynamic light scattering (DLS) is based on the Brownian motion of dispersed particles. When particles are dispersed in a liquid they move randomly in all directions. The principle of Brownian motion is that particles are constantly colliding with solvent molecules. These collisions cause a certain amount of energy to be transferred, which induces particle movement. The energy transfer is more or less constant and therefore has a greater effect on smaller particles. As a result, smaller particles are moving at higher speeds than larger particles. By knowing all other parameters which have an influence on particle movement, one can determine the hydrodynamic diameter by measuring the speed of the particles.

          The relation between the speed of the particles and the particle size is given by the Stokes-Einstein equation (Equation 1). The speed of the particles is given by the translational diffusion coefficient DA. Further, the equation includes the viscosity of the dispersant and the temperature because both parameters directly influence particle movement. A basic requirement for the Stokes-Einstein equation is that the movement of the particles needs to be solely based on Brownian motion. If there is sedimentation, there is no random movement, which would lead to inaccurate results. Therefore, the onset of sedimentation indicates the upper size limit for DLS measurements. In contrast, the lower size limit is defined by the signal-to-noise ratio. Small particles do not scatter much light, which leads to an insufficient measurement signal.

          DA=KT6πηRA\displaystyle D_A=\frac{KT}{6{\mathrm{πηR}}_{\mathrm A}}

          The basic setup of a DLS instrument is shown in Figure 3. A single frequency laser is directed to the sample contained in a cuvette. If there are particles in the sample, the incident laser light gets scattered in all directions. The scattered light is detected at a certain angle over time and this signal is used to determine the diffusion coefficient and the particle size by the Stokes-Einstein equation.

          Intensity Trace and Correlation Function

          The scattered light is detected over a certain time period in order to monitor the movement of the particles. The intensity of the scattered light is not constant but will fluctuate over time. Smaller particles, which are moving at higher speeds, show faster fluctuations than larger particles. On the other hand, larger particles result in higher amplitudes between the maximum and minimum scattering intensities. This initial intensity trace is further used to generate a correlation function. In general, the correlation function describes how long a particle is located at the same spot within the sample. At the beginning the correlation function is linear and almost constant, indicating that the particle is still at the same position as it was the moment before. Later there is an exponential decay of the correlation function, which means that the particle is moving. If there is no similarity to the initial spot, the correlation function shows a linear behaviour again. This part of the correlation function is known as the baseline. The information of the size-dependent movement is included in the decay of the correlation function. The decay represents an indirect measure of the time that the particles need to change their relative positions. Small particles move quickly so the decay is fast. Larger particles move more slowly and therefore the decay of the correlation function is delayed.

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            Autocorrelation graph

            Auto correlation function

            To understand the autocorrelation of scattered intensity, consider a time varying signal, I(t) monitored at different intervals of time. If we represent the scattered intensity at an arbitrary time as I(t) and those after a delay time τ as I( t + τ), the normalized auto correlation function of the scattered intensity, K(τ)can be written as

            K(τ)=I(t)I(t+τ)\displaystyle K(\tau)=\left\langle I(t)I(t+\tau)\right\rangle

            Experimentally, the average designated by the brackets in above equation is a time average over the data collection interval T

            K(τ)=1T0TI(t)I(t+τ)dτ\displaystyle K(\tau)=\frac1T\int_0^TI(t)I(t+\tau)d\tau

            We, define a quantity, autocorrelation function

            g2(τ)=I(t)I(t+τ)I(t)2\displaystyle g^2(\tau)=\frac{\left\langle I(t)I(t+\tau)\right\rangle}{\left\langle I(t)\right\rangle^2}

            Where,

            I(t)=1T0TI(t)dt\displaystyle \left\langle I(t)\right\rangle=\frac1T\int_0^TI(t)dt

            When the delay time τ is zero, the signal is perfectly correlated and the un-normalized correlation function) in above expression yields a value of ⟨I2⟩.When τ is infinity, it is perfectly uncorrelated yielding a value of ⟨I⟩2.From statistics, one can see that ⟨I2⟩ is greater than or equal to ⟨I⟩2.The decay of this function follows a characteristic time scale depending on the diffusion coefficient of the scatterers. Autocorrelation function of scattered light corresponds to single exponential decay.

            Small Angle Neutron Scattering Techniques.

            Small-angle neutron scattering (SANS) is an experimental technique that uses elastic neutron scattering at small scattering angles to investigate the structure of various substances. Small angle neutron scattering measurements can provide structural details of materials on a scale covering a range from 1 nm to few hundred nm. SANS arises from variations of scattering length density. Details of the porosity and surface area can be obtained from measurements of the angular distribution of the scattered intensity.

            In SANS experiments, one measures the coherent scattering intensity as a function of Q which can be given by

            I(Q)=NV2(ρpρs)2P(Q)S(Q)+B\displaystyle \begin{array}{l}I(Q)=NV^2(\rho_p-\rho_s)^2P(Q)S(Q)+B\\\end{array}

            where N is the number density of particles and V is particle volume. ρP and ρS are scattering length densities of particle and solvent, respectively, wave vector transfer Q =(4πsinθ)/λ; 2θ is scattering angle, λ is wave length of neutrons. P(Q) is the form factor, which describes the size and shape of the scattering objects and S(Q) is interparticle structure factor. If the system of scatterers has no interparticle correlation (e.g. it is a dilute solution) then S(Q) = 1. This is a valid approximation for the system i.e. polymer nanoparticles. B is the incoherent background coming from the hydrogenous contents. For spherical particle of radius r, P(Q) can be given as

            P(Q)=[3(Sin(Qr)QrCos(Qr))2(Qr)3]\displaystyle P(Q)=\left[\frac{3(Sin(Qr)-QrCos(Qr))^2}{(Qr)^3}\right]

            The SANS technique

            The neutrons produced are passed through a monochromator to make all neutrons have the same wavelength. Then it passes through the collimator which making all the neutrons parallel. Thus the collimated beam of neutrons is hits on a sample and scattered by the dispersed particles in the sample. The scattered beam of neutrons is detected by a detector.

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              SANS instrument in DHRUVA reactor

              SANS Facility at DHRUVA

              The small-angle neutron scattering experiments presented in this report are performed at SANS diffractometer, Dhruva reactor, BARC Mumbai. The schematic representation of SANS diffractometer installed at the guide tube laboratory of Dhruva reactor is shown in Scheme 1 and Scheme 2. The neutron beam from the guide is monochromatized using a velocity selector. The velocity selector selects the particular velocity neutrons using multi-slotted multi-discs rotating at high speed (rpm = 4000-7000). The mean wavelength and wavelength spread of the monochromatic beam can be varied in the range 4 to 10 Å and 8 to 20%, respectively. This beam passes through two slits S1 (2 cm × 3 cm) and S2 (1 cm × 1.5 cm) before it reaches the sample. Distance between S1 and S2 is 2 m and gives an angular divergence of 0.5º. The angular distribution of neutrons scattered by the sample is recorded using a one-dimensional position sensitive detector. The sample to detector distance is 1.85 m. The Q range of the diffractometer is 0.015-0.35 Å-1.

              Screenshot (3).png
                SANS facility at DHRUVA

                Results and Discussion

                Among the numerous preparation methods, solvent displacement method is considered as a convenient method for the preparation of polymer nanoparticles. The addition of polymer to a magnetically stirred aqueous solution confirms the formation of polymer nanoparticles. The size of the polymer nanoparticle depends on many factors. An investigation is followed to study the factors that influence the size of the nanoparticles.

                Results

                Solute concentration variation

                In order to study the effect of solute concentration on the size of the nanoparticles, different concentration of PCL in water is made. The samples were made from 5% PCL to 60% PCL in water.0.5 ml of 3mg/ml concentration of PCL is added to calculated volumes of water to prepare the samples. The samples analysed by DLS measurements. It is found that the size of the polymer nanoparticles is increasing when goes to higher concentration.

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                  Auto correlation of samples with increasing concentration

                  The information of the size-dependent movement is included in the decay of the correlation function. The decay represents an indirect measure of the time that the particles need to change their relative positions. Small particles move quickly so the decay is fast. Larger particles move more slowly and therefore the decay of the correlation function is delayed. Viscosity is affected the hydrodynamic size. Hence viscosity corrections are made in the size.

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                    Size of particles without viscosity measuremen

                    Dilution of samples

                    It is found that size of the particles when diluting higher concentration to 5 times, larger size is decreasing to small. The change in size of nanoparticles is related to the change in viscosity of nanoparticles.

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                      Size of the particles diluted to 5 times.

                      Influence of Solvent in the size of polymer nanoparticles

                      The size of the polymer nanoparticles are influenced by the amount of acetone in the polymer colloid. The size dependence of polymer nanoparticles on acetone is studied by comparing the size effect with a reference solution. The reference solution is prepared by adding 1.2 ml PCL in acetone to 4 ml of water and the sample is characterised by DLS. The size of the polymer nanoparticles in the reference solution was observed as 240.28 nm.

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                        auto correlation of Reference solution

                        Taking 2 ml of reference solution and adding 0.1 ml acetone to it for studying its size change. The sample after prepared was characterised by DLS technique. The acetone volume is increased successively in the reference sample. It is observed that the size of nanoparticles gradually increased with increasing the content of additional acetone in the solution.

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                          Autocorrelation function of nanoparticles when solvent volume increased.

                          Influence of Solvent to Non- solvent ratio in the size of nanoparticles

                          The ratio of water to acetone influences the size of the nanoparticles. When the solvent to non-solvent ratio changed, the size of PNP’s is also changed. The total volume of sample is made at 4.5 ml and 90% of 4.5 ml is set to be the volume of water and remaining is solvent which is added to the aqueous phase. 0.5 ml of PCL in acetone (3 mg/ml) is added to this solution and size analysed using DLS technique. Similarly for 80% water in 20% acetone, 70% water in 30% acetone, etc. up to 50% water in 50% acetone in aqueous phase is prepared and samples were analysed.

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                            Autocorrelation function of nanoparticles at different water to acetone ratio

                            Influence of Salt on polymer particle size

                            NaCl is known to solubilise in water rapidly while they are insoluble in acetone. The solubility of NaCl is decreased in the mixture of water and acetone, and hence some of the dissolved salt will precipitate out. This will change the diffusion coefficient of the solution and the size and stability of nanoparticles.

                            0.1ml PCL (3mg/ml) in different molar concentration of salt

                            The size of the polymer nanoparticles are influenced by NaCl solution. This is done by making samples of different concentrations of NaCl in water and adding PCL in acetone (3mg/ml) to it. Different concentration of NaCl solution was prepared and 0.1 ml of 3mg/ml concentration of PCL is added to it.

                            For 0.1 ml PCL solution added to different molar concentrations of NaCl solution it is to be noted that at higher molar concentration particle size is found to be larger.

                            From this graph it is clear that at higher concentrations, the particle size is very large and for lower molar concentration size became smaller. The process is made to be reverse. That is, after prepared the reference solution in water, adding molar concentrations of NaCl solution to it to study the size effect of salt on reference sample. 0.1 ml of PCL solution added to 1 ml of water and reference solution was prepared to which salt solutions of suitable volume are added. For reference solution the size was observed 177.06 nm. The size distribution is given below

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                              autocorrelation of reference solution

                              When adding salt to the reference solution, the particle size is increased to higher molar concentration.

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                                auto correlation graph of salt added to reference solution

                                0.2 ml PCL in different molar concentration of salt

                                0.2 ml of PCL in 1 ml water is prepared (reference solution). For the Reference solution the size obtained as 207.22 nm. 0.2 ml PCL in acetone (3mg/ml) is added to different molar concentrations of salt and the size variations was studied.

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                                  autocorrelation of samples when PCL is added to different concentrations of salt.

                                  0.3 ml PCL in different molar concentration of salt

                                  0.3 ml in 1 ml of water is made and studied the effect of salt on polymer nanoparticles. Here also the particles size increased at higher molar concentration exactly same as above. The size of polymer particles in the reference sample was found to be 224.94 nm.

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                                    Auto correlation of reference sample

                                    Adding 0.3 ml PCL solution to various molar concentrations of NaCl samples, at lower concentration size is small, but when going to higher size become larger. This variation in size is shown below,

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                                      Auto correlation of samples when 0.3 ml PCL added to salt

                                      Effect of Salt on PLGA Nanoparticles.

                                      Instead of using PCL in Acetone(3 mg/ml), Another polymer in Acetone at higher concentration was selected. 5 mg/ml concentrations of PLGA solution is prepared. A reference solution of PLGA in water is made by adding 0.25 ml of PLGA in 1 ml of water. For the reference solution, it is noted that the size of the particle is 132 nm.

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                                        Auto correlation of reference solution

                                        Also different molar concentration of PLGA (0.25 ml) in Nacl solution is prepared.The size for the particles when adding PLGA in salt is increasing at higher molar concentration.

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                                          Auto correlation PLGA in different concentrations of salt

                                          The size of the polymer nanoparticles in the reference solution is smaller as compared to PLGA in higher concentrations. To study the size variations, salt is added to the reference solution and it is observed that the size is increasing slightly.

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                                            Auto correlation graph of salt added to reference.

                                            Effect of Urea on the size of Polymer nanoparticles

                                            Thus, Sodium Chloride solution as aqueous phase considerably increases the size of the polymer particles at higher concentrations. Similar size effect was studied using UREA as aqueous phase.

                                            A reference solution is prepared by adding 0.25 ml PLGA in 1 ml water and size of the particles was observed as 148.52 nm.

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                                              Auto correlation of Reference sample

                                              0.25 ml of PLGA solution was added to 1 ml of different concentrated urea solutions in water. It was noted that unlike salt, urea will not alter the size of the polymer nanoparticles. The auto correlation graph is given below,

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                                                Auto correlation of PLGA in Urea

                                                Discussions

                                                In this investigation, it is found that as concentration of the sample increases, the size of the particle is also increases (Fig.1). This can be explained the synergetic effect of solute induced growth and the interaction of solvent-nonsolvent and solvent-polymer interaction parameter. When the polymer solution is injected water, instantaneously nanoparticles are formed due to the velocity of diffusion of acetone towards water. The growth of nanoparticles instantaneously occurred with increasing the polymer concentration. In addition, the increasing content of acetone reduces the polarity of the solvent and encourages the growth of nanoparticles. At higher acetone concentration, the coalescence takes place together with the particle growth.

                                                However, with dilution of the concentrated suspension to 5 times, it is found that the size of particles is decreasing and comparable to the size of 5% PCL in water. When we add water to the polymer colloid, the polarity of the solvent recovered and the particles become stable at smaller size. The aggregation of the particle is basically reduces if we diluting the solutions.

                                                The volume of Acetone also plays a vital role in the formation as well as size controlling of PNP’s. It was found that the size of the polymer nanoparticles is increasing when the amount of acetone is increased. It is known that the size of the polymer nanoparticles more strongly correlated to the solvent-water interaction parameter compared to the solvent water mutual solubility parameter. The solvent-water interaction parameter solvent-water is expressed as

                                                χSolventWater=VSolventRT(δSolventδWater)2\displaystyle \chi_{Solvent-Water=\frac{V_{Solvent}}{RT}\left(\delta_{Solvent}-\delta_{Water}\right)^2}^{}

                                                where V is the molar volume of the organic solvent, R is the gas constant, T is the temperature, and δsolvent and δwater are the Hildebrand solubility parameters of solvent and water, respectively. According to the above expression, the size of nanoparticles increased significantly with increasing acetone content, which in accordance with the solvent-water mutual solubilization theory.

                                                Amount of acetone and water also influences the size of the PNP’s. This was confirmed by changing both the parameters simultaneously. The sample information is given below,

                                                sample information of experiment
                                                sample

                                                90%water

                                                +10%acetone

                                                (s1)

                                                80%water+

                                                20% acetone

                                                (s2)

                                                70%water+

                                                30%acetone

                                                (s3)

                                                60%water+

                                                40%acetone

                                                (s4)

                                                50%water+50%acetone(s5)

                                                ACETONE(gm)

                                                8.1568

                                                8.1568

                                                6.062

                                                8.1568

                                                6.062

                                                POLYMER (gm)

                                                0.0303

                                                0.0303

                                                0.0303

                                                0.0303

                                                0.0303

                                                INITIAL CONCENTRATION(g/ml)

                                                0.00291

                                                0.00291

                                                0.00291

                                                0.00291

                                                0.00291

                                                Total volume of sample

                                                4.5

                                                4.5

                                                4.5

                                                4.5

                                                4.5

                                                water in sample(gm)

                                                4.02

                                                3.583

                                                3.1791

                                                2.6747

                                                2.251

                                                acetone in sample(gm)

                                                0.36

                                                0.6428

                                                1.3629

                                                1.3332

                                                2.25

                                                mass of PCL in acetone(gm)

                                                0.3909

                                                0.394

                                                0.3919

                                                0.3914

                                                0.3971

                                                mass of PCL in final sample(gm)

                                                1.449

                                                1.4609

                                                1.5879

                                                1.4512

                                                1.609

                                                Final concentration (mg/ml)

                                                0.291

                                                0.298

                                                0.293

                                                0.298

                                                0.286

                                                Influence of salt on the size of PNPs

                                                Effect of salt on PNP’s are investigated and it is verified by preparing different molar concentration of salt and 0.1 ml of PCL (3mg/ml) is added to these samples. The size of the polymer nanoparticles considerably increased for larger molar concentrations shown in the Fig. 9.The sample information aregiven below,

                                                sample information of experiment

                                                sample

                                                PCL in 50mM saltPCL in 20mM saltPCL in 10mM saltPCL in 5mM saltPCL in 3mM salt

                                                POLYMER (gm)

                                                0.0245

                                                0.0245

                                                0.0245

                                                0.0245

                                                0.0245

                                                ACETONE(gm)

                                                6.062

                                                6.062

                                                6.062

                                                6.062

                                                6.062

                                                INITIAL CONCENTRATION(g/ml)

                                                0.00318

                                                0.00318

                                                0.00318

                                                0.00318

                                                0.00318

                                                Mass of solution(gm)

                                                0.9972

                                                0.9964

                                                0.9954

                                                1.0006

                                                0.9962

                                                mass of PCL in acetone(gm)

                                                0.0745

                                                0.0766

                                                0.0778

                                                0.0722

                                                0.0747

                                                mass of PCL in final sample(gm)

                                                0.00030218

                                                0.0003107

                                                0.00031557

                                                0.00029285

                                                0.00030299

                                                final concentration (mg/ml)

                                                0.277

                                                0.284

                                                0.288

                                                0.268

                                                0.278

                                                To understand the effect of salt on PCL NP’s, a reference solution is prepared in water (Fig.10) salt solution of suitable volume is added to the reference solution and it was observed that size is increasing (Fig. 11).

                                                For 0.2 ml PCL, it is also observed that size of PCL particles were increasing at higher concentrations of salt (Fig. 13).The sample information is given below,

                                                sample information of 0.2 ml PCL in salt

                                                sample

                                                PCL in 100mMPCL in 50mM PCL in 20mM PCL in 10mM PCL in 5mM PCL in 3mM

                                                POLYMER (gm)

                                                0.0245

                                                0.0245

                                                0.0245

                                                0.0245

                                                0.0245

                                                0.0245

                                                ACETONE(gm)

                                                6.062

                                                6.062

                                                6.062

                                                6.062

                                                6.062

                                                6.062

                                                INITIAL CONCENTRATION(g/ml)

                                                0.00318

                                                0.00318

                                                0.00318

                                                0.00318

                                                0.00318

                                                0.00318

                                                Mass of solution(gm)

                                                1.0041

                                                0.9962

                                                0.9977

                                                0.9995

                                                0.9998

                                                1.0005

                                                mass of PCL in acetone(gm)

                                                0.1556

                                                0.1571

                                                0.1603

                                                0.1556

                                                0.1556

                                                0.1561

                                                mass of PCL in final sample(gm)

                                                0.00063

                                                0.00064

                                                0.00065

                                                0.0006310.000630.000633

                                                final concentration (mg/ml)

                                                0.525

                                                0.533

                                                0.541

                                                0.527

                                                0.527

                                                0.528

                                                Size of PCL particles related to salt is shown in the Fig. 15.The sample information is given below,

                                                sample information of 0.3ml PCL in salt

                                                sample

                                                PCL in 100mMPCL in 50mMPCL in 20mMPCL in 10mMPCL in 5mMPCL in 3mM

                                                POLYMER (gm)

                                                0.0304

                                                0.0304

                                                0.0304

                                                0.0304

                                                0.0304

                                                0.0304

                                                ACETONE(gm)

                                                8.0535

                                                8.0535

                                                8.0535

                                                8.0535

                                                8.0535

                                                8.0535

                                                INITIAL CONCENTRATION(g/ml)

                                                0.00296

                                                0.00296

                                                0.00296

                                                0.00296

                                                0.00296

                                                0.00296

                                                Mass of solution(gm)

                                                0.9978

                                                1.041

                                                0.9969

                                                0.9989

                                                0.9955

                                                0.9991

                                                mass of PCL in acetone(gm)

                                                0.2411

                                                0.2698

                                                0.2398

                                                0.2371

                                                0.2411

                                                0.2402

                                                mass of PCL in final sample(gm)

                                                0.00091028

                                                0.00101863

                                                0.00090537

                                                0.00089517

                                                0.00091028

                                                0.00090688

                                                final concentration (mg/ml)

                                                0.697

                                                0.735

                                                0.695

                                                0.688

                                                0.699

                                                0.695

                                                5 mg/ml PLGA in acetone was prepared and 0.25 ml of PLGA added to salt solutions of suitable volume, here also similar results obtained as explained above. This is shown inFig. 17.A reference sample is prepared in which suitable volume of NaCl solution added and size of PLGA particles is observed as increasing (Fig. 18). The sample information is given below,

                                                Sample information of PLGA in salt

                                                sample

                                                PLGA in 0.8mMPLGA in 0.9mMPLGA in 1mMPLGA in 3mMPLGA in 5mM

                                                PLGA

                                                In

                                                10mM

                                                PLGA

                                                in 20mM

                                                50

                                                mM

                                                100

                                                mM

                                                POLYMER (gm)

                                                0.0273

                                                0.0273

                                                0.0273

                                                0.0273

                                                0.0273

                                                0.0273

                                                0.0273

                                                0.0273

                                                0.0273

                                                ACETONE(gm)

                                                4.284

                                                4.284

                                                4.284

                                                4.284

                                                4.284

                                                4.284

                                                4.284

                                                4.284

                                                4.284

                                                INITIAL CONCENTRATION(g/ml)

                                                0.004996

                                                0.00499

                                                0.0049

                                                0.00499

                                                0.0049

                                                0.0049

                                                0.00499

                                                0.00499

                                                0.00499

                                                Mass of solution(gm)

                                                1.7957

                                                1.8937

                                                1.9496

                                                1.9939

                                                1.9819

                                                1.979

                                                1.973

                                                1.9748

                                                1.984

                                                mass of PCL in acetone(gm)

                                                0.1991

                                                0.2006

                                                0.1913

                                                0.192

                                                0.1988

                                                0.1982

                                                0.1983

                                                0.1984

                                                0.189

                                                mass of PCL in final sample(gm)

                                                0.00126877

                                                0.00127833

                                                0.00121783

                                                0.00122228

                                                0.00126557

                                                0.00126175

                                                0.00126239

                                                0.00126302

                                                0.00120318

                                                final concentration (mg/ml)

                                                0.562

                                                0.567

                                                0.537

                                                0.542

                                                0.564

                                                0.564

                                                0.563

                                                0.563

                                                0.538

                                                NaCl is known to solubilise in water rapidly while they are insoluble in acetone. The solubility of NaCl is decreased in the mixture of water and acetone, and hence some of the dissolved salt will precipitate out. This will change the diffusion coefficient of the solution and the size and stability of nanoparticles.

                                                The Reason for the increase in size of the polymer nanoparticles can be explained in terms of Zeta potential measurements. Zeta potential is a measure of particle stability in the dispersion medium or it is a measure of degree of repulsion between the particles. When the zeta potential is high (more negative or positive), the particles in the medium is stable. When the zeta potential is lower, the attractive wan der wall attraction exceeds the repulsion, hence aggregation or coalesces takes place.

                                                When the polymer nanoparticles dissolved in acetone is added to the salt solutions of higher molar concentration, the zeta potential is found to be smaller. The solubility NaCl is decreased in the mixture of water and acetone, and hence some of the dissolved salt will precipitate out. As a result, the polarity of the solvent decreased rapidly and the attractive force become dominants over the repulsive one. Thus the particle aggregate to form larger nanoparticles. As the concentration of the salt decreases, the zeta potential is found to be larger. Thus, they will not aggregate and remains in the stable state due to high repulsion. Thus, higher molar concentration, the hydrodynamic size is larger. 0.25 ml PLGA is added in 1 ml water and whose zeta potential is found to be -54 mv.

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                                                  Zeta potential measurements of reference solution

                                                  But, when adding PCL (3mg/ml) to the salt solution it is found that zeta potential is decreasing.

                                                  On the contrary, the addition of urea has no effect in these two polymer nanopaerticles formation. Unlike NaCl, urea has significant solubility in acetone. Therefore, the addition of urea does not change the solvent polarity and remains in the solution as part of nanoprecipitation. That is why the final size of the nanoparticles remains unchanged even at higher fraction (2M) of urea in the solution.

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                                                    Zeta potential of PLGA in salts
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                                                      SANS study of polymer nanoparticles at different salt concentrations.

                                                      Some of the selected nanoparticles in the final state are measured by SANS (Fig. 24). The size of the nanoparticles is too big to resolve the complete form factor. However, the Porod Scattering is observed in the high-q range. The intensity is decreased by q-2.9 for the nanoparticles without salt and 2.2 for the nanoparticles containing 50 mM NaCl, reveals the formation of surface fractal [6]. The shifting of SANS intensity towards lower-q for the nanoparticles contains 50 mM NaCl suggest the formation of bigger structure compared to the without salt system. The SANS results are in well agreement with the observation made from DLS results.

                                                      Zeta potential value of PLGA in salt
                                                      Sample NameZeta Potential(mV)
                                                      PLGA in 10mM salt-45.7
                                                      PLGA in 20mM salt-33.3
                                                      PLGA in 30mM salt-26
                                                      PLGA in 50mm salt-22.9
                                                      PLGA in 100mM salt-10.9
                                                      PLGA in 200mM salt-9.8mV

                                                      Conclusion

                                                      Polymer nanoparticles formation through nanoprecipitation results from the dilution of the solute molecules in water, causing from the nucleation of very small aggregates, subsequent growth of solute molecules, which is followed by the coalescence phenomenon. The size and polydispersity of polymer nanoparticles obtained via nanoprecipitation method can be controlled by the final concentration of solute and solvent condition. The presence of salt plays an important role in defining the final size and stability of nanoparticles.

                                                      References

                                                      [1] M. Gaumet, A. Vargas, R. Gurny and F. Delie, Eur. J. Pharm. Biopharm., 2008, 69, 1–9.

                                                      [2] S. M. Moghimi, Target. Drugs Deliv. Syst., 1995, 17, 61–73.

                                                      [3] M. Beck-Broichsitter, E. Rytting, T. Lebhardt, X. Wang and T. Kissel, Eur. J. Pharm. Sci., 2010, 41, 244–253.

                                                      [4] J.G.J.L.Lebouille, R.Stepanyan, J.J.M.Slotb, M.A.Cohen Stuart, R.Tuiniera, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2014, 225-235.

                                                      [5] Philippe Legrand, Sylviane Lesieur, Amélie Bochot, Ruxandra Gref, Wouter Raatjes, Gillian Barratt, Christine Vauthier, International Journal of Pharmaceutics, 2007, 33-43.

                                                      [6] Katsuji Noda, Mitsuhisa Ohashi, Kiyoharu Ishida, J. Chem. Eng. Data, 1982, 27, 326-328.

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