International Journal of Pharmaceutics

Table 1. summarises the pros and cons of choosing ... a high thermodynamic stability and smaller particle size, ... Databases and search strategy...

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International Journal of Pharmaceutics 533 (2017) 275–284

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Review Article

Technical aspects of preparing PEG-PLGA nanoparticles as carrier for chemotherapeutic agents by nanoprecipitation method Hassan A. Almoustafaa, Mohammed A. Alshawsha, Zamri Chika,b, a b

MARK



Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia University of Malaya Bioequivalence and Testing Centre (UBAT), Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

A R T I C L E I N F O

A B S T R A C T

Keywords: Nanoprecipitation PEG-PLGA Ouzo effect Nanoparticles Biodegradable polymers Nanoparticles

Nanoprecipitation is a simple and increasingly trending method for nanoparticles preparation. The self-assembly feature of poly (ethylene glycol)-poly (lactide-co-glycolic acid) (PEG-PLGA) amphiphilic copolymer into a nanoparticle and its versatile structure makes nanoprecipitation one of the best methods for its preparation. The aim of this study is to review currently available literature for standard preparation of PEG-PLGA nanoparticles using nanoprecipitation technique in order to draw conclusive evidenceto draw conclusive evidence that can guide researchers during formulation development. To achieve this, three databases (Web of Science, Scopus and PubMed) were searched using relevant keywords and the extracted articles were reviewed based on defined inclusion and exclusion criteria. Data extraction and narrative analysis of the obtained literature was performed when appropriate, along with our laboratory observations to support those claims wherever necessary. As a result of this analysis, reports that matched our criteria conformed to the general facts about nanoprecipitation techniques such as simplicity in procedure, low surfactants requirement, narrow size distribution, and low resulting concentrations. However, these reports showed interesting advantages for using PEG-PLGA as they are frequently reported to be freezedried and active pharmaceutical ingredients (APIs) with low hydrophobicity were reported to successfully be encapsulated in the particles.

1. Introduction Nanoprecipitation was first reported by (Fessi et al., 1989). They used the term “interfacial deposition” to describe their method in which a mixture of Poly (lactic acid) (PLA), benzyl-benzoate and phospholipids in hot acetone was used to encapsulate indomethacin (a hydrophobic compound) and the mixture was added dropwise into water with poloxamer as a surfactant. The solvent was then evaporated under reduced pressure. The resulting particles were described as a capsule consists of a film of mainly PLA and a core of benzyl-benzoate containing the drug. Nanoprecipitation is the simplest laboratory based polymeric nanoparticle preparation method ever steadily reported to date. In an optimum case of an amphiphilic polymer based particles prepared using this method; preparation does not require high sheer homogenization techniques, ultracentrifugation or surfactants. However, this method is hindered by significant drawbacks such as; low concentration preparation, the arguably inability to freeze dry the particles (Bodmeier et al., 1991). Table 1. summarises the pros and cons of choosing



nanoprecipitation to prepare PEG-PLGA nanoparticles. As the research in nanomedicine grows, the trends in publications of nanoprecipitation and nanoparticles has continued going up especially in the last five years as presented in Fig. 1. On top of the difficulties that conventional chemotherapeutics should pass through to get approved by regulatory agencies worldwide which make it exceptionally expensive, nanotechnology also involves several additional special manufacturing steps, requiring elongated time for batch preparation and specially trained personnel. The process usually lacks robustness and reproducibility, and may result in continuous shortage of supplies even for the oldest formulations in the market (Barenholz, 2012; Food and Administration, 2015). For this reason, simplicity of nanoprecipitation is anticipated to reduce batch to batch variabilities and ensure consistency of the manufacturing process. Zhu, (2013) Answered the debate about block copolymer nanoparticles formed by flash nanoprecipitation proposing that they are an accumulation of polymer structures that were kinetically frozen in a non-equilibrium thermodynamic state, hence they are neither micelles nor micellar particles that are composed of clearly identified layers with

Corresponding author. E-mail address: [email protected] (Z. Chik).

http://dx.doi.org/10.1016/j.ijpharm.2017.09.054 Received 8 May 2017; Received in revised form 17 September 2017; Accepted 18 September 2017 Available online 22 September 2017 0378-5173/ © 2017 Elsevier B.V. All rights reserved.

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Table 1 Advantages and disadvantages of nanoprecipitation method to prepare nanoparticles from amphiphilic polymers. Characteristic Water miscible solvents

Spontaneous (dropwise) particle formation No surfactant Needed

Low energy mixing

Advantages

Disadvantages

toxic (nonhalogenated) (Sah and Sah, 2015) • Less • Able to encapsulate less hydrophobic drugs.

solely on the ouzo effect which limits the polymers that can be used. • Depends solvent removal causes particle aggregation hence they are more • Incomplete difficult to be freeze dried or collected by ultracentrifugation. to encapsulate hydrophilic drugs using simple nanoprecipitation • Difficult techniques. concentration of the dispersed phase (high concentration will result in • Low an uneven and large particle distribution).

single step process for hydrophobic drug • Simple encapsulation. • Narrowly dispersed particle size. toxicity of surfactants. • Avoid surface characteristics effects. • Avoid centrifugation speed is enough to collect the • Lower particles with high yield high sheer homogenization, making scaling up • No easier Francois and Katz (2005).

system can aggregate over period of time. • Metastable • Sedimentation and aggregation can occur and affect physical stability.

• Improper mixing can disturb particle size distribution.

Fig. 1. Trends and comparison of publications of nanoprecipitation and nanoparticles. *Data was extracted from Web of Science.

Fig. 2 shows the phase diagram of a system composed of a hydrophobic solute, polar miscible solvent and water, namely single phase, nucleation and separation phase. A single-phase region is just a stable solution (lowest water and salute concentrations), while nucleation

a high thermodynamic stability and smaller particle size, with some of the PEG chains trapped inside the hydrophobic core and some sticking out of the particle to form the hydrophilic surface. Fig. 3 illustrates the difference between a nanoparticle and a micelle.

Fig. 2. Phase diagram of ternary system: the nucleation area is determined by the red colour and bordered by the spinodal and the bimodal lines. The system is monodispersed and best metastable in the Ouzo region. Adopted with slight modification from (Botet, 2012). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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formation methods were always identified when mentioned. If the above-mentioned criteria were met, research articles were still can be excluded if the formulation was prepared using double emulsion, or if a high sheer homogenization method like sonication was applied or when the solvent was not a water miscible.

region according to classical nucleation theory (CNT) starts when the two-phase system evolves by nucleation. This region is delimited by the binodal line with the single-phase region and the spinodal line with the phase separation region. The best metastable state in this region is achieved in a small portion of it called the Ouzu region in which the particles are narrowly dispersed in the aqueous solution (Botet, 2012). Separation phase region happens when the system is thermodynamically stable again after complete separation and two phases can be observed macroscopically. In CNT these three phases of particle formation are driven by supersaturation which occurs upon adding the organic phase (undersaturated organic solution) to the aqueous phase. This will produce a supersaturated solution as the hydrophobic salute (block copolymer) is not soluble in the new mixture. This process is thought to be affected by two factors. First is the value of supersaturation that is determined by the solute concentration; the higher it is, the bigger nuclei are formed. The second factor is the interfacial tension between the salute and the water solvent, and therefore, mixing plays a critical role in the size distribution where slow mixing results in big sized low number of nuclei with bigger size and bad size distribution (Botet and Roger, 2016; Lepeltier et al., 2014). Choosing PEG-PLGA over other amphiphilic block-copolymers such as polystyrene block polyethylene glycole (PS-PEG) and polycabrolactone block polyethylene glycole (PCL-PEG) was done because of its superior physical and chemical characters like biodegradability, glass transition temperature above room temperature, non-crystallinity, low affinity with PEG makes the PEG chain always on the outer side of the particulates formed during ouzo process and stabilize the core shell structure, PLGA is also approved by US FDA for parenteral administration (Zhu, 2013). In this review we are focusing on parameters that considered as important obstacles and most critical steps in the process of PEG-PLGA nanoparticles preparation using nanoprecipitation technique, which include; polymer selection, organic phase, aqueous phase, drug encapsulation and freeze drying.

2.3. Data extraction Selected studies were searched and critically summarised for the details of technical aspects necessary for preparation of PEG-PLGA nanoparticles using nanoprecipitation technique. These technical aspects considered as important obstacles and the most critical steps in the process of PEG-PLGA nanoparticles preparation, which include; polymer selection, organic phase, aqueous phase, drug encapsulation and freeze-drying steps. 3. Critical technical steps in preparing PEG-PLGA nanoparticles by nanoprecipitation 3.1. Polymer selection When choosing PLA, PLGA polymer, inherent viscosity (IV) is the basic measure given by manufacturers; it has different patterns by changing the molecular weight (MW) and depending on the ratio between lactic and glycolic acid. PLGA chain terminals can be either left as carboxylic group that exploited with esterification reactions to connect with other compounds (small, macro molecules or polymers), or capped with esterification (with alkyl or methyl esters) which are more stable, and have lower degradation rates (Houchin and Topp, 2008). On the other hand, poly (glycolic acid) (PGA) is highly crystalline with high melting point (225–230 °C) and low solubility in organic solvents (depends on the MW). PLA is a chiral molecule and is amorphous in its racemic mixture, namely, poly-DL-lactic acid (PDLLA). The more of the L enantiomer the more the degree of crystallinity, and since this enantiomer occurs naturally in the body, it is more biocompatible (Athanasiou et al., 1996). For PLGA, the more glycolide content the faster degradation of the copolymer, except in case of 50:50 ratio of glycolic and lactic that is reported to have the fastest degradation (Miller et al., 1977; Park et al., 1995). Pegylation products are now available with several shapes and end groups. The end groups of the PEG determine the structure of the final polymer. Chemically inert group doesn’t allow additional coating of the particle while adding excess amount of PEG with reactive end groups should be enough to allow the formation of di-block copolymer with free groups to react with targeting moieties on the surface of these particles. Fig. 3 depicts how PEG and PLGA polymers are configured depending on the block copolymer structure depending on the preparation method. It illustrates the difference between a nanoparticle kinetically formed by nanoprecipitation and a micelle slowly thermodynamically assembled. Nanoprecipitation of the amphiphilic copolymer is considered a favourable method to form pegylated PLGA nanoparticles as it provides high stability and high coverage density (Rabanel et al., 2014). The length of each of the copolymer chains plays a significant role in the water solubility of the block copolymer. The bigger the ratio of the PEG to the PLGA, the more likely for it dissolve in water (Akina, 2015). However, the size of the resulting particles may not be significantly affected by the length of the two chains, as the particle formation mechanism could be through spinodal decomposition (Horn and Rieger, 2001) and the size can be independent from the ratio of the diblocks in some ranges (Zhu et al., 2007). However, this solubility may vary depending on the termination groups. There is no point in using a soluble copolymer, and applying law temperature conditions during the copolymer purification and nanoparticles preparation, which means can be proposed to get proper

2. Methodology 2.1. Databases and search strategy Three databases were searched for research articles, namely PubMed, Scopus and Web of Science. The following keywords using Boolean logic were considered for gathering the relevant literature (pegylated PLGA nanoparticles cancer) OR (pegylated PLA nanoparticles cancer) OR TOPIC: (PEG-PLGA nanoparticles cancer) OR (PEG-PLA nanoparticles cancer) OR (PEG-PLGA nanoprecipitation) OR (PEG-PLA nanoprecipitation) OR (pegylated PLGA nanoprecipitation) OR (pegylated PLA nanoprecipitation) OR (PEG-b-PLGA nanoparticles cancer) OR (PEG-b-PLA nanoparticles cancer) OR (PEG-b-PLGA nanoprecipitation) OR (PEG-b-PLA nanoprecipitation). Articles were screened based on prespecified inclusion and exclusion criteria on two stages. Primary screening was conducted based on (title, abstract and Keywords). Then; the full text of the selected articles was critically analysed and included for synthesis of this review. 2.2. Inclusion and exclusion criteria Published articles in English were considered if they contained a detailed explanation of nanoparticle formulation such as the PEG-PLGA or PEG-PLA di or tri block amphiphile were prepared prior to the formulation process. Studies were considered only if more than 50% of the amphiphile is not chemically attached to any ligand, small molecules or macromolecules, solvents have to be water miscible and was used as a carrier for chemotherapeutics. Studies of all types of encapsulated entities, automated nanoprecipitation and micelle formation by dialysis methods were included in the synthesis of this review. Micelle 277

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Fig. 3. Structure of tri-block copolymer (A): micelle, di-block copolymer (B) micelle (C): tri-block copolymer nanoparticle (D): di-block copolymer nanoparticle, PLGA: Poly lactic-coglycolic acid, PEG: Polyethylene glycol.

Table 2 Basic characteristics of possible water miscible solvents (Reichardt, 2004), (ICH, 2011). Solvent

Acetone Acetonitrile THF DMF DMSO Methanol Ethanol Water

Boiling point (°C)

56.2 81.6 66 153 189 64.5 78.3 100

Vapor pressure (hPa)in 20 °C

PDE (mg/day)

240 97 200 3.5 0.61 128 59 17.5

– 4.1 7.2 8.8 – 30 – –

Concentration limit (ppm)

– 410 720 880 – 3000 – –

Table 3 List of solvents or solvents combination frequently used for nanoprecipitation based on the retrieved studies.

ICH classa

III II II II III II III –

a ICH: International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use solvent classification: Class I: Solvents that should be avoided, Class II: Solvents to be limited to the stated levels, Class III: Solvents with low toxic potential. 50 mg per day or less (corresponding to 5000 ppm) would be acceptable without justification. Higher levels can be accepted if they are realistic with regard to GMP guidelines. PDE: Permissible Daily Exposure, THF: t, DMF: dimethylformamide, DMSO: dimethyl sulfoxide.

Solvent

Frequency

Acetone THF DMSO DMF Acetonitrile PEG DMSO + acetone (1:4 v/v ratio) Acetonitrile + DMSO Acetone or THF Acetone + ethanol (60:40 v/v) Acetone, THF, DMF or DMAC Acetone, THF, Acetone + DMF Acetone, DMF, DMSO, THF, DMSO + THF (1:1) or DMSO + Acetone (1:1,1:4)

22 9 8 8 5 1 2 1 1 1 1 1 1

THF: tetrahydrofuran, DMF: dimethylformamide, DMSO: dimethyl sulfoxide, PEG: polyethylene glycol, DMAC: dimethyl acetamide.

chemistry) or purchased as a readymade from manufacturers. However, two interesting reports can be commented on; Li et al., has synthesized dumbbell shaped copolymers (PLGA)2PEG(PLGA)2, which showed lower critical micelle concentration (CMC), smaller particle size, higher

yield of some high hydrophilic to hydrophobic ratio copolymers. Polymers in the retrieved studies either were prepared and characterized in conventional ways (ring opening polymerization, EDC-NHS 278

279

Micelle formation by Dialysis.

Daman et al. (2014)

Stearoyl gemcitabine

Doxorubicin

Rhodamine B

Docetaxel

Doxorubicin

Doxorubicin

all-trans-retinoic acid

Empty with bigger NPs reported when encapsulated flourouracil and Paclitaxel

Paclitaxel

Encapsulated compound

PEG5000-PLA4500 PEG5000-PLA16000 PEG5000-PLA23000 PLA3000-PEG2000PLA3000 PLA3000-PEG4000PLA3000 mPEG2000-PLA2000

0.0046 0.0070 0.0028 0.00225 0.02 0.2 0.01122 0.00374 ± 23

210 ± 0.5 168.8 ± 1.3 375.6 442.1 302.3 295.8 374.7 178.1

0.014 0.0316

43 22.44 22.86

81.95 195 83.45 110

0.024 0.023 0.076 0.080 LogCMC 2.1 0.00794 Empty Log-2.65 = 0.00224 PTX 0.02649 0.04848 0.0724 0.017

34 43 107 107 180 empty 210 PTX

± 69

± 41.6 ± 36.1 ± 56.8

0.0042

138.0 ± 0.5

0.00178

CMC (g/l)

0.0024

89.9 ± 4.73

PEG5000PLA5100COOH PLA61-PEG91-PLA61 MW 12710 PLA68-PEG91-PLA68 MW13710 PLA90-PEG91-PLA90 MW17940 PLA62-PEG182-PLA62 MW16900 mPEG5000-PLA5000 mPEG5000-PLA10000 mPEG5000-PLA20000 mPEG2000-PLA16000 mPEG2000-PLA32000 PLGA-PEG-PLGA 122400 by NMR 21300 by GPC PEG2000-PLA3000 PEG5000-PLA3000 PEG2000-PLA5000 PEG5000-PLA5000 PLA-PEG-PLA

b

63.8 ± 3

Size (nm)

Formulation

The polymer has a double disulfide linkage

paclitaxel increased hydrophobicity and resulted in lower CMC

Used linear and dumbbell shaped polymers, the result shown here is for the lineara

Reported critical association concentration for polymeric micelles instead of CMC calculated from mol/l

Reported the measurement of critical flocculation point in which a dramatic change in turbidity appears. Flocculation was induced by adding an electrolyte

Note

a Dumbbell shaped polymers achieved lower CMC and higher EE for Doxorubicin., PEG: Polyethylen glycol, PLA: Polylactic Acid, mPEG: Methoxypolyethylene glycol, PLGA Polylactic co glycoic acid, THF: Tetrahydrofuran, NMR: Nuclear magnetic resonance, GPC: Gel permeation chromatography, DMSO: Dimethyl sulfoxide. b CMC in all the investigated articles was determined by fluorescence spectroscopy using pyrene as a fluorescent probe.

Yang et al. (2016)

THF Acetone THF + Acetone DMSO slowly added to water then dialyzed against water

Sezgin-bayindir et al. (2016)

THF dropped on PBS then dialysed

Li et al. (2013)

In THF then water was added

Acetone volatilization dialysis (evaporation for 0.5 h then dialysis for 24 h

Li et al. (2009)

Xie et al. (2007)

Nanoprecipitation then solvent removal Acetone and THF evaporated, DMF and DMAC removed by dialysis

Venkatraman et al. (2005)

In DMSO into water then dialysed

Dialysis

Xu et al. (2009)

Yang et al. (2015)

Preparation Method

Reference

Table 4 Summary of critical micelle concentration (CMC) measurements.

H.A. Almoustafa et al.

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3.2. Organic phase

Table 5 List of surfactants used for nanoprecipitation. Surfactant (w/v)

Choosing an appropriate solvent and selecting the most suitable solvent removal techniques are critical steps in nanoparticles preparation. Solvents removal will be discussed in the context of freeze-drying. Other parameters such as solvent to water ratio were discussed previously (Miladi et al., 2016). Choosing the solvent is the basic step in nanoprecipitation size tuning and encapsulation efficiency. shows a list of water miscible solvents and some of their physicochemical properties. Acetone is the best solvent available for nanoprecipitation as it is less toxic and easy to be removed due to low boiling point. Two methods are commonly used in solvent removals which are evaporation and dialysis. To ensure good removal of solvent by evaporation, the solvent evaporation temperature should be significantly lower than water (solvent vapour pressure should be higher than water) (Table 2). However, solvent removal rate has been proven to have no significant effect on particles characteristics (Chorny et al., 2002). On laboratory scale, vacuum is commonly used to accelerate solvent evaporation, besides time saving, which can add to the reproducibility, physical and chemical stability of the formulation. Although dialysis is inefficient and time consuming scaled up (Saad and Prud’homme, 2016), dialysis is still being used extensively because some solvents such as DMF and DMSO cannot be removed by evaporation. It should be noted that an increasing in time during this step can make particles prone to aggregation. To avoid this, special measures should be taken such as adding a surfactant or using special commercially available dialysis kits to ensure continuous spinning of the particles. Table 3 summarises the solvents or solvents combination which are frequently used for nanoprecipitation according to the retrieved articles

Reference

In Aqueous Phase Tween-80 (0.2%) Tween-80 (0.1, 0.25 or 0.5%) PVA (2.5%) Poloxamer 188 (2%)

Lin et al. (2013) Bonaccorso et al. (2017) Poojari et al. (2015, 2016) Chaudhari et al., (2012); Yadav et al. (2010) Lee et al. (2010)

Poloxamer 407 (0.1%) In organic phase Lipoid S75, 66 mg/mL of acetone (soybean phospholipid) Macrogol 15 Hydroxystearate (0.5%) Dual Stabilization Tween 80, Macrogol 15 Hydroxystearate soybean lecithin 25 mg in 5 ml of organic phase, Tween 80 (0.2%) Polyvinylpyrrolidone (0.2%) No Surfactant

Nguyen et al. (2008) Guo et al. (2015) Karra et al. (2013) Klippstein et al. (2015) Tam et al. (2016) Rest of the articles

PVA: Polyvinyl alcohol. PVP: Polyvinylpyrrolidone.

drug loading and also demonstrated slower in vitro drug release (Li et al., 2013). In another study (PEG)3 PLA copolymer was prepared for the sake of increasing pegylation density, it was reported that this polymer slowed the hydrophilic drug release from the self-assembled micelles (Ayen et al., 2011).

Table 6 Summary of drug loading of the hydrophobic drug Docetaxel in PEG-PLGA copolymer using nanoprecipitation. Docetaxel: water solubility: 0.0127 g/La LogP 2.4 Reference

Solvent

Surfactant

Dmg/Pmg/ Oml/Wmlb

Polymer

Size

EE%c

DL%d

Chaudhari et al. (2012) Grover et al., 2014 Liu et al. (2010)

Acetone

7.5/100/Xe/ 20 ml 4/120/3/10

PEG3350PLGA12000 mPEG5000-PLGA

132

73.53

5.51

Acetone

Poloxamer 188 (0.5% w/v) none

27.70

Acetone

none

X/10 mg/ 5 ml/5 ml

PEG% PLGA

Acetone

none

10 mg/ 0.5 mg/ 1 ml/2 ml

PLA-TPGS/TPGSCOOH (80:20)

374.25 83.76f 162.7 190.7 225.4 202.5 192.7 ≈180 for nontargeted

Zhao and Feng (2014)

Le et al., 2016

Acetone

none

Acetone Bonaccorso et al. (2017)

Zhang et al. (2014)

1 mg/ 10 mg/1:5 0.9/30/8/16

Tween 80 (0.1; 0.25 or 0.5% w/V), also “surfactant-free” Dimethyl sulfoxide (DMF)

None

10/100/10/ 15

PEG-PLGA later reacted with scFv PEG5% PEG10% PEG15%

PEG-PLGA 10000

a

0 5 10 15 20 2000 3350 5000 2000 3350 5000

≈210 for targeted

105

Size depend on the degree of pegylation when it is high or the surfactant concentration when its low 38.3

34.1 7.1 7.28 9.87 5.42 5.55 5.11 Na

280

NA

Increased Pegylation reduced loading capacity of hydrophobic drugs 72.8

From https://www.drugbank.ca/drugs. D: Drug concentration in organic solvent (mg), P: copolymer concentration in organic solvent (mg), O: organic solvent (ml), W: aqueous phase (ml). c EE: encapsulation efficiency. d DL drug loading. e Not mentioned. f Aggregated and non-aggregated sizes. PLGA: Poly lactic-co-glycolic acid, PEG: Polyethylene glycol. b

NA

7.1

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Table 7 Summary of drug loading of the hydrophilic drug Doxorubicin in PEG-PLGA copolymer using nanoprecipitation. Doxorubicin (water solubility: 1.18 g/L)a LogP: 1,42 Reference

Solvent

Surfactant

D/P/O/W

Polymer

Size

EE%

DL%

Lee et al. (2010)

Acetone

2/50/6/50

PEG-PLGA

99.1

NA

3.9

Lu et al. (2011)

DMSO

Poloxamer 407 (0.1% w/v) none

NA

NA

21

Ayen and Kumar (2012a)

none

4/15/1.5/5

171.3

51.7

10.9

none

NA

(PEG)3-PLA 17.5 KDa

133.9

NA

NA

Li et al. (2013)

Acetone/ DMSO 4/1 Acetone/ DMSO 4/1 THF

mPEG5000-PLGA1510 (multifunctional) (PEG)3-PLA 17.5 KDa

none

10/50/5/2

DMSO

none

1/10/1/8

Tsai et al. (2010)

DMF (dialysis)

none

6.25 7.97 8.35 2.59 2.79 4.65 3.20 9.8–21.2

Song et al. (2016)

DMSO

none

20/30/10/(0;10;20;30;40;50%) (dialysis) 3/10/5/(dialysis)

178.1 97.9 38.8 34 43 107 107 200–250 nm

33.33 43.3 45.55

Yang et al. (2015)

Yang et al. (2016)

DMSO

none

1/10/1/8

Tam et al. (2016)

DMF

none 0.2% PVP none 0.2% PVP

2/10/1/19

PLGA-PEG-PLGA 21300 (PLGA)2-PEG-(PLGA)2 20000 (PLGA)2-PEG-(PLGA)2 34400 PEG2000-PLA3000 PEG5000-PLA3000 PEG2000-PLA5000 PEG5000-PLA5000 PEG5000-PLA and (HEMA-cohistidine)-g-PLA PEG-PLA PEG-PLA-PEG PLA3000-PEG2000-PLA3000 PLA3000-PEG4000-PLA3000 PEG2000-PLA10000

Ayen and Kumar (2012b)

Acetone

a

323 ± 47.9 106 ± 2.2 110 43 90.5 83,2 53.4 49.8

5.05 4.73 48.6 43.4 36.1 32.6

From https://www.drugbank.ca/drugs, D: Drug concentration in organic solvent mg, P: copolymer concentration in organic solvent mg, O: organic solvent ml, W: aqueous phase ml.

Table 8 Ability of common solvents used in nanoprecipitation to be freeze-dried, adopted with slight modification from (Sprung, 2012). Solvent

Freezing point (°C)

Collector required (°C)

Additional notes

Acetone Acetonitrile DMSO Ethanol Methanol Methylene Chloride Salt water Water

−94.3 −42 18.5 −117.3 −97.8 −97 Varies on PPM 0

−105 −84 Cascade −50 −105 −105 −105 −84 Cascade −50

Must be diluted to 15% Acetone DMSO can be difficult to freeze dry Must be diluted to 15% ethanol Must be diluted to 15% methanol Must be diluted to 15% methylene chloride PTFE-coated chamber recommended

along with the amount of surfactant and concluded that the lower the PEG in copolymer formula the more the surfactant amount, which plays a role in controlling the particle size.

3.3. Aqueous phase Nano sized particulate solutions that possess a low CMC value are speculated to have a longer circulation time and are more stable thermodynamically (Torchilin et al., 2003). High CMC value can result in the breakdown of the micelles in the circulation (Trivedi and Kompella, 2010). It is interesting to see all this literature measuring CMC values for nanoprecipitation preparations, seven of the 8 screened articles followed a nanoprecipitation procedure and used CMC measurement. Table 4 summarises CMC measurements obtained in the selected studies. This review shows that majority of studies that comply with our criteria did not use a surfactant at all besides the PEG-PLGA amphiphile. This indicates efficiency of the amphiphilic characteristics of PEG-PLGA copolymer. Only few studies have reported the usage of both water soluble and oil soluble surfactants. Two studies have used a double stabilizing system (water soluble and oil soluble surfactants) in both aqueous and organic phases (see Table 5.). In contrary to general assumptions about stability of nanoprecipitation formulations without surfactants. Table 5 summarizes the used surfactants in addition to the PEG-PLGA block copolymer in the screened articles. (Bonaccorso et al., 2017) has optimized the degree of pegylation

3.4. Drug encapsulation The scientific principle behind increasing encapsulation efficiency is to inhibit drug diffusion during solvent removal. This can be achieved by tweaking several parameters; namely increasing the particle size and copolymer concentration in the organic phase, selecting a solvent with a higher affinity to the drug, adjusting the copolymer composition (PEG to PLGA and lactide to glycolide) and altering the pH to reduce water solubility (Miladi et al., 2016). Zhu et al. introduced n-octanol/water partition coefficient (LogP) as an indicator for drug hydrophobicity, they proved that it is better correlated with particle stability comparing to solubility parameter (δ). and they concluded that particles with an APIs that has a logP less than 2 were unlikely to form while APIs with logP ranging between 2 and 9 showed fast Ostwald ripening and recrystallization. To form a stable particle we need logP values bigger than 12 (Zhu, 2014). Interestingly, the screened literature showed compounds with low LogP to be the most commonly used with PEGPLGA. 281

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Table 9 Summary of freeze-drying protocols used for nanoparticle preparation. Referencea,b

Solvent removal

Stabilizer

Particle collection

Cry/lyoprotectantc

Ayen and Kumar (2012) Chaudhari et al. (2012) Guo et al. (2015)

Dialysis Evaporation Evaporation

None Poloxamer Kolliphor 15 SH

None Centrifugation 0.45 mm cellulose acetate filter

Liu et al. (2010) Gao et al. (2013) Jung et al. (2015) Li et al. (2013) Xu et al. (2009) Asadi et al. (2011) Lee et al. (2010) Li et al. (2009) Lin et al. (2013) Lu et al. (2011) Mu et al. (2013) Sun et al. (2015) Tsai et al. (2010) Venkatraman et al. (2005) Shalaby et al. (2014) Voruganti et al. (2015) Lale et al. (2015) Ma et al. (2015) Zhang et al. (2014) Tam et al. (2016) Pustulka et al. (2013)

Evaporation Evaporation at 60 °C Evaporation at 37 °C and vacuum

None None None None None None Pluronic F-127 (0.1% w/v None 0.2% Tween-80 None None None None None None 0.5% PVA None None None None or PVPd 0.2% None None None None

Centrifugation Not mentioned Centrifugation Filtration Filtration Centrifugation Centrifugation Filtration Centrifugation with water None Filtration Filtration Not mentioned None Centrifugation Centrifugation None None None None None Ultracentrifugation Ultra centrifugal filter None

Inulin Trehalose Trehalose 5% (w/v) 3% w/w sucrose None None None None None None None None None None None None None None None 10% trehalose None None None None None None None

Zhou et al. (2015) Han et al. (2015) a b c d

Dialysis Evaporation Evaporation Dialysis Evaporation under 60 ° then centrifugational washing Dialysis Evaporation under vacuum Evaporation Dialysis (micelle formation) Evaporation for acetone THF and dialysis for DMF DMAC Evaporation Evaporation Dialysis Micelles (thin film hydration) Dialysis NA not removed NA Evaporation not removed

Freeze-drying for characterization purposes only that are not meant to keep the integrity of the particles or were done to the copolymer before particle formation are neglected. Copolymer differences may be taken into consideration also. The details are not reported. PVP: Polyvinylpyrrolidone.

cause aggregation of the particles during lyophilisation, this residues are estimated by the partition coefficient of methanol in water to that of the organic phase(Lepeltier et al., 2014). However, some of the retrieved studies have used freeze dried during nanoparticle formulations (Bodmeier et al., 1991; Francois and Katz, 2005). The organic phase choice substantially affects the nanoformulations ability to be freeze dried. Table 8. indicates that conventional freezedriers are not able to remove most of the solvents that used in nanoprecipitation. Although acetone evaporates from water mixtures at low temperature, its residue cannot be removed by normal freeze-drying machine, thus the particles are expected to aggregate resulting in inability to be suspended again at the same particle size distribution. (Tong et al., 2010) have optimized lyoprotection for nanoparticles that match our criteria and they found that bovine serum albumin protected the particle characteristics and saved their ability to be reconstituted maintaining their targeting capabilities. Other freeze-drying procedures have reported the use of other cryoprotectants, but most of the screened articles did not mention any (Table 9). This may lead to the conclusion that freeze-drying PEG-PLGA nanoparticles prepared by nanoprecipitation is now a common successful practice due to frequency of reports.

3.4.1. Hydrophobic drugs Nanoprecipitation is usually used to encapsulate hydrophobic compounds. Encapsulation of a drug depends on its partition coefficient between the hydrophobic core of the particle and the aqueous phase. High chemical compatibility between the frequently used (API) and the polymer can be estimated using Flory-Huggins solution parameters (Zandanel and Charrueau, 2016). The most frequent example of hydrophobic drugs is Docetaxel. Table 6 summarises the Docetaxel encapsulation in all existing formulations. 3.4.2. Hydrophilic drugs One of the main disadvantages of nanoprecipitation is that it has low encapsulation efficiency for hydrophilic compounds. Several attempts to overcome this problem have been reported, among the proposed solutions is controlling the solubility of the encapsulated drug using pH adjustment (Govender et al., 1999). Another way is through designing the drug itself to suit the formulation either by chemical manipulation (Wang et al., 2014) or by releasing the compound from its hydrophilic salt form (often more chemically stable than the hydrophobic compound). It should be noted that salt formation are simpler than covalent modifications as it does not require full re-approval from FDA (Pinkerton et al., 2013). A common example of this is doxorubicin HCl which is constantly reported to be mixed with triethylamine to increase its hydrophobicity, hence improve its encapsulation efficiency (Yang et al., 2015). In fact, anthracyclines are considered the best hydrophilic drugs to be encapsulated using nanoprecipitation. Table 7 demonstrates the technical details of doxorubicin loaded nanoparticles formulations according to the retrieved articles.

4. Conclusion To summarize this review, it should be emphasized that PLGA and PEG selection plays a key role in particle characteristics. Micelle formulations using dialysis or thin film hydration were found to be reported as nonoprecipitation preparations and nanoprecipitation formulations were also described as micelles. CMC measurement should be considered irrelevant to nanoprecipitation kinetically stabilised formulations. It is worth noting that surfactants become important especially when low PEG content is used, hence the amphiphilic characteristics of the particles are compromised and need to be

3.5. Freeze drying It is hypothesised that the residues of water miscible solvent can 282

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