Scale-independent microfluidic production of cationic liposomal

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Scale-independent microfluidic production of cationic liposomal adjuvants and development of enhanced lymphatic targeting strategies. Carla B Roces, Swapnil Khadke, Dennis Christensen, and Yvonne Perrie Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00730 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 25, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Molecular Pharmaceutics

Scale-independent manufacture of liposomal adjuvants

Proven efficacy  Biodistribution

Lymphatic targeting

Avidin pre-dose

 Antibody responses  Cytokine responses Biotin-decorated liposomes

ACS Paragon Plus Environment

Lymph nodes

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Title:

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Page 2 of 42

Scale-independent microfluidic production of cationic liposomal adjuvants and development of enhanced lymphatic targeting strategies.

3 4

Authors:

Carla B. Roces1, Swapnil Khadke1, Dennis Christensen2 and Yvonne Perrie1*

5

1

6

Glasgow, Scotland.

7

2

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde,

Center for Vaccine Research, Statens Serum Institut, Copenhagen, Denmark.

8 9 10 11

*Corresponding author:

12

Professor Yvonne Perrie

13

Strathclyde Institute of Pharmacy and Biomedical Sciences,

14

161 Cathedral St,

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University of Strathclyde,

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Glasgow, G4 0RE

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Scotland.

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[email protected]

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1 ACS Paragon Plus Environment

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Abstract

22

Cationic liposomes prepared from dimethyldioctadecylammonium bromide (DDAB) and trehalose

23

6,6′-dibehenate (TDB) are strong liposomal adjuvants. As with many liposome formulations, within

24

the laboratory DDAB:TDB is commonly prepared by the thin film method which is difficult to scale and

25

gives high batch-to-batch variability. In contrast, controllable technologies such as microfluidics offer

26

robust, continuous and scale-independent production. Therefore, within this study, we have

27

developed a microfluidic production method for cationic liposomal adjuvants that is scale-

28

independent and produces liposomal adjuvants with analogous biodistribution and immunogenicity

29

compared to those produced by the small-scale lipid hydration method. Subsequently, we further

30

developed the DDAB:TDB adjuvant system to include a lymphatic targeting strategy using

31

microfluidics. By exploiting a biotin-avidin complexation strategy, we were able to manipulate the

32

pharmacokinetic profile and enhance targeting and retention of DDAB:TDB and antigen within the

33

lymph nodes. Interestingly, re-directing these cationic liposomal adjuvants did not translate into

34

notably improved vaccine efficacy.

35 36 37

Key words: microfluidics, manufacture, vaccine adjuvants, cationic liposomes, lymphatic targeting.



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1. Introduction

39

Liposomes have been extensively studied as vaccines adjuvants. However current production methods

40

for liposomes are costly, multi-step and generally limited to batch production. Given that the cost of

41

vaccines is a key contributing factor in global accessibility, low-cost scalable production of vaccine

42

adjuvants is required to ensure an affordable supply chain. To address this and bring down the costs

43

of liposomal adjuvants, streamlining their manufacturing process is essential. Recently, the application

44

of microfluidics has been demonstrated for a range of nanoparticles and liposomes (e.g.

45

Microfluidics as a manufacturing platform offers a scale-independent alternative to batch production

46

and the nanoparticle product attributes have been shown to be process controlled in terms of particle

47

size and high protein loading can be achieved compared to other production methods 1.

1-5

).

48 49

In the manufacture of liposomal adjuvants, control of the physicochemical attributes is vital given that

50

these are often critical quality attributes. Indeed, a range physicochemical attributes have been shown

51

to impact on the immunological properties of liposomal adjuvants including particle size 6-9, charge 10-

52

12

53

physico-chemical attributes also dictate the pharmacokinetic properties of both the liposomal

54

adjuvant and the sub-unit antigen and the recruitment of antigen presenting cells (APCs) to the

55

injection site 8. Thus by modifying these attributes, both the pharmacokinetic and immunogenic

56

profile can be manipulated. In particular, the use of cationic lipids has a strong impact on both the

57

adjuvanticity of liposomes and their retention at the site of injection. For example, the use of the

58

cationic lipid N,N;’dimethyl-N,N’-dioctadecylammonium bromide (DDAB) has been shown to favour

59

the absorption of subunit antigens onto the liposomal surface, promote retention of both the adjuvant

60

and antigen at the injection site and promote strong cell mediated immune responses 10. To formulate

61

liposomal adjuvants, DDAB is often used in combination with the synthetic immunopotentiator α,α‘-

62

trehalose-6,6’-dibehenate (TDB) to improve the stability of the liposomes and enhance the

63

immunogenicity of the formulation 22. TDB is a synthetic analogue of trehalose 6,6’-dimycolate (TDM),

, lipid composition 11, 13, 14, fluidity 14-17 and degree of pegylation 18-21. Furthermore, several of these


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a mycolic acid from the mycobacterial cell wall from Mycobacterium tuberculosis and it is combined

65

with DDAB at a weight ratio of 5:1 (DDAB:TDB)22. The adjuvanticity of DDAB:TDB is generated via the

66

Syk – Card9 – Bcl10 – Malt1 pathway. By this way, TDB activates macrophages and dendritic cells (DCs)

67

23

68

macrophages) which also stimulates MyD88-dependent Th1/Th17 responses may contribute to

69

DDAB:TDB efficacy

70

immune responses based on high IFN-ɣ and IL-17 secretion, low IL-5 production and high IgG antibody

71

production

72

investigated to consider potential links between biodistribution and vaccine efficacy. By dual

73

radiolabelling of the adjuvant and the antigen, it has been shown that cationic liposomes form a depot

74

at the injection site, followed by a sustained release to the draining lymph nodes 15, 28. However, it has

75

also been shown that direct injection of DDAB:TDB into the lymph node promotes a strong response

76

29

77

liposomal formulation; whilst no significant differences were found between subcutaneous,

78

intramuscular or intradermal vaccination, intra-lymphatic administration of DDAB:TDB liposomes

79

resulted in significantly higher IgG2a and IFN-ɣ responses

80

increased dose of DDAB:TDB to draining lymphatics and further enhance immune responses is an

81

interesting consideration. To promote retention at the draining lymphatics, a biotin-avidin complex

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formulation can be adopted. Studies carried out by Phillips et al. 30 demonstrated the ability of this

83

high affinity complex to improve the accumulation and retention of liposomes into the draining lymph

84

nodes. By this means injection of biotin-coated liposomes, in combination with an adjacent

85

intramuscular injection of avidin, become localised/trapped in the draining lymph nodes due to the

86

formation of avidin-biotin-coated liposome complexes in the lymphatics 30, 31. Thus, exploiting the high

87

affinity between biotin and avidin resulted in higher accumulation of liposomes in the lymph nodes

88

(up to 14%) in comparison to biotin-coated liposomes injected without avidin (2% of the injected dose)

89

30

. Moreover, interaction with the Mincle receptor (a C-type lectin receptor expressed in

24, 25

. Through this pathway, DDAB:TDB promotes strong cellular and humoral

26, 27

. The pharmacokinetic profile of DDAB:TDB and numerous variants has also been

. Indeed, Mohanan et al. showed the importance of the route of administration for the DDAB:TDB

29

. Thus, the potential to re-direct an

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As with many liposome formulations, the common method for preparing DDAB:TDB liposomal

92

adjuvants within the laboratory is via the hydration method (LH) 22, 32 which results in the formation

93

of a large multilamellar vesicles (MLVs) which are heterogeneous in nature. In order to reduce the size

94

and lamellarity of these liposomes, sonication or high shear mixing can be applied. This produces small

95

unilamellar vesicles (SUVs) and in the case of DDAB:TDB this can reduce the particle size down from

96

approximately 500 nm to 200 nm and a polydispersity (PDI) between 0.2 and 0.4 17, 32. However, these

97

processes are difficult to scale-up and are generally limited to small scale laboratory production.

98

Therefore, to address the need for scale-independent manufacture of cationic liposomal adjuvants we

99

have investigated the use of microfluidics for the production of DDAB:TDB. We have then applied this

100

method to develop a modified biotinylated DDAB:TDB formulation that can promote liposome and

101

antigen drainage to, and retention within, the lymphatics in order to test the impact this has on

102

vaccine efficacy.

103 104

2. Materials and methods

105

2.1 Materials

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The cationic surfactant dimethyldioctadecylammonium (DDAB) bromide, the immunopotentiator

107

trehalose

108

[biotinyl(polyethylene glycol)-2000] (DSPE-PEG(2000) biotin) were purchased from Avanti Polar Lipids

109

Inc. (Alabaster, USA). Avidin (egg white) and cholesterol were purchased from Sigma-Aldrich Company

110

Ltd. (Poole, UK). Hybrid 56 (H56) tuberculosis vaccine candidate was gifted by Statens Serum Institut

111

(Copenhagen, Denmark). Tris-base was obtained from IDN Biomedical Inc. (Aurora, OH, USA) and used

112

to make 10 mM Tris buffer and adjusted to pH 7.4 using HCl. The radionucleotides Iodine 125I (NaI in

113

NaOH solution) and Tritium 3H-cholesterol (tritium-labelled cholesterol in ethanol), and Ultima Gold™

114

scintillation fluid were purchased from Perkin Elmer (Waltham, MA, USA). Sodium hydroxide (NaOH)

115

and hydrogen peroxide (H2O2) were purchased from Sigma-Aldrich Company Ltd. (Poole, UK).

6,6′-dibehenate

(TDB)

and

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-


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Bicinchoninic acid protein assay (BCA) kit and Sephadex G-75 superfine were purchased from Fisher

117

Scientific (Leicestershire, UK). IODO-GEN® pre-coated iodination tubes from Pierce Biotechnology

118

(Rockford, IL) and scintillation vials from Sardsted Ltd (Leicester, UK). Horseradish peroxidase (HRP)

119

enzyme (HRP-streptavidin), purified rat anti mouse IFN-ɣ and IL-17, biotin conjugates IFN-ɣ and IL-17,

120

IL-17 standard, and mouse IL-5 ELISA set were purchased from Becton Dickinson (BD biosciences, New

121

Jersey, US). Mercaptoethanol, concanavalinA (conA), Tween 20, Bovine serum albumin (BSA),

122

carbonate-bicarbonate buffer tablets, sulfuric acid, IFN-ɣ standard, skimmed milk powder, heparin,

123

bovine serum albumin, sodium chloride (NaCl), sodium azide (NaN3), Triton X-100 and protease

124

inhibitor

125

Tetramethylbenzidine (TMB) substrate, isotype-specific immunoglobulins (Goat anti-mouse IgG1 and

126

IgG2c), Penicillin-Streptomycine (10,000 U/mL), L-glutamine 200 mM, sodium pyruvate 100 mM, MEM

127

non-essential amino acids solution (100x), RPMI 1640 media, Fetal Bovine Serum (FBS), HEPES (1 M),

128

Phosphate Buffered Saline (10x) were purchased from Fisher Scientific - UK Ltd (Loughborough, UK).

cocktail

were

purchased

from

Sigma-Aldrich

Company

Ltd.

(Poole,

UK).

129 130

2.2 Manufacture of liposomes.

131

Two techniques for the production of liposomes were applied and compared: LH and microfluidics.

132

For the LH method, liposomes were prepared by a modification of the Bangham method 33. Briefly,

133

lipid stocks of DDAB and TDB were dissolved in a mixture of chloroform and methanol (9:1 v/v). The

134

required amount of lipid solution was transferred to a round bottom flask to reach the appropriate

135

final concentration (5 mg/mL DDA and 1 mg/mL TDB). Organic solvent was removed under vacuum

136

with a rotary evaporator during 15 min at 200 revolutions per minute (rpm). The lipid film was

137

hydrated with the desired amount of 10 mM Tris buffer (pH 7.4) at 60°C for 20 - 30 min.

138 139

The preparation of liposomes by microfluidics was conducted on the Nanoassemblr® Benchtop system

140

from Precision Nanosystems Inc. Stocks of DDAB and TDB were prepared in 2-propanol (IPA) and

141

mixed to the desired concentration (in general 20 mg/mL of DDAB and 2mg/mL TDB). Selected speeds 6 ACS Paragon Plus Environment


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(total flow rates (TFRs)) and ratios between the aqueous and organic phase (flow rate ratios (FRRs))

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were investigated with FRRs of 1:1, 3:1 and 5:1 (solvent to aqueous phase) and TFRs of 5, 10 and 15

144

mL/min tested. During the process, samples were heated to ensure the lipids stayed dissolved and the

145

heating block was set at 60°C. To prepare biotinylated liposomes, DSPE-PEG(2000) biotin was added

146

to the DDAB:TDB formulation at a 20 mol% ratio in order to investigate the effect of the biotin-avidin

147

complex in the distribution of these particulate systems within the body. Concentrations ranging

148

primarily between 0.3 – 24 mg/mL total lipid were tested. H56 antigen (5 µg per vaccine dose (50 µL))

149

was mixed with preformed liposomes after production. Solvent was removed by dialysis (dialysis

150

tubing Mw 12,000-14,000 Da, Sigma Aldrich, Poole, UK) against Tris buffer. For free-antigen removal

151

a 300,000 Da MWCO membrane was used (Spectra-Por®, Spectrum Labs, Breda, The Netherlands).

152 153

2.3 Determination of the particle size, PDI and zeta potential.

154

The size of liposomes was determined by dynamic light scattering (Zetasizer nano ZS, Malvern

155

PANalytical Ltd., Worcestershire, UK). Samples manufactured using the LH were measured

156

approximately one hour after preparation to allow samples to cool down, whereas the samples

157

manufactured using microfluidics were measured directly after purification. For zeta potential

158

measurement, samples were diluted in the same fashion as for the size. Three measurements of each

159

sample at 25°C were taken.

160 161

2.5 Quantification of lipid recovery.

162

Quantification of the lipid recovery was performed by high performance liquid chromatography (HPLC,

163

YL Instruments Co. Ltd. Korea) using an SEDEX 90LTD ELSD detector (Sedex Sedere, Alfortville, France)

164

as described previously 34. A Luna 5 µ C18(2) column (Phenomenex, Cheshire, UK), pore size of 100Å,

165

was used. Lipids were dissolved in chloroform:methanol (9:1 v/v) and liposomal samples were injected

166

without preparation. HPLC-ELSD settings were kept constant as follows: 30 µL injection volume in a

167

partial loopfill injection mode, 100 µL loop volume and 15 µL tubing volume. Column temperature was 7 ACS Paragon Plus Environment

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maintained at 35°C whereas the ELSD temperature was set at 52°C in all the runs. Nitrogen was used

169

as a carrier gas at 3.5 psi inlet pressure. Clarity DataApex version 4.0.3.876 was used for data analysis.

170 171

2.6 Quantification of antigen loading.

172

Quantification of the antigen loading on the liposomal formulations was performed by reverse phase

173

HPLC (RP-HPLC) using a UV detector (Agilent Technologies, Edinburgh, UK) as described previously 35.

174

A Jupiter 5 µ C18(2) column (Phenomenex, Cheshire, UK) pore size 300Å was used as stationary phase.

175

For the preparation of the standards and samples, antigen alone or liposomes loaded with H56, were

176

diluted in 50% Tris/IPA (1:1 v/v) 36. Mobile phase A contained 90% H20, 10% acetonitrile and 0.1% TFA

177

whereas Mobile phase B contained 70% acetonitrile, 30% H20 and 0.1% TFA. The instrument settings

178

were as follows: 50 µL injection volume, flow rate 1 mL/min, UV wavelength 210 nm and column

179

temperature 60°C.

180 181

2.7 Stability of liposomes in simulated in vivo conditions.

182

Stability of the liposomal formulations was assessed in terms of size, PDI and zeta potential under

183

simulated in vivo conditions. Briefly, liposomes were placed in a water bath at 37°C with 50% FBS and

184

aliquots were taken at specific time points in line with the biodistribution time points.

185 186

2.8 In vivo studies.

187

All in vivo studies were conducted under the regulations of the Directive 2010/63/EU. All protocols

188

have been subject to ethical review and were carried out in a designated establishment. During all

189

studies, mice were weighted weekly and examined to detect any significant change in their health. All

190

mice had a healthy weight, characteristic of the strain and age, with no significant differences between

191

groups.

192 193

2.8.1 Biodistribution studies of liposomal adjuvants and their associated antigen. 8 ACS Paragon Plus Environment


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Inbred female BALB/C mice (3 mice per time point) were obtained from the Biological Procedure Unit

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at the University of Strathclyde, Glasgow. All mice used were 6-10 weeks of age at the start of the

196

experiment. Liposomal formulations were radiolabelled with 3H-Cholesterol by incorporation of the

197

isotope into the lipid bilayer 37. For isotonicity, 10% w/v trehalose was added to the filtered buffer

198

(0.22 µm filter). Radiolabelled H56 antigen was added to the hydrated formulation at the desired

199

concentration (0.1 mg/mL) for surface loading onto the DDAB:TDB formulation. This formulation was

200

used as a control since its biodistribution vaccine efficacy has been reported previously 8, 10, 11, 13, 18, 38.

201

All liposomal formulations contained a final concentration of 250 µg DDAB/50 µg TDB/5 µg H56 per

202

vaccine dose (50 µL). In order to investigate the effect of the biotin-avidin complex in the distribution

203

of these DDAB:TDB liposomes, DSPE-PEG(2000) biotin was added to the DDAB:TDB formulation at a

204

20 mol% ratio. When necessary, avidin (200 µg per dose) was injected intramuscularly 2 h prior main

205

immunization with the biotinylated formulation in the same quadriceps (adjacent injections) 30.

206 207

Mice were injected intramuscularly with 50 µL of the radiolabelled formulation in the right quadriceps

208

and mice were terminated after 6, 24, 48, 96 and 192 hours. Specific organs/tissues were isolated:

209

inguinal lymph node (ILN), mesenteric lymph node (MLN), popliteal lymph node (POP), spleen and site

210

of injection. These organs/tissues were added in a scintillation vial containing 2 mL NaOH 10 mM for

211

dissolution. Mice carcasses were dissolved with 60 mL of NaOH 10 mM for mass balance calculation.

212

All vials containing NaOH were incubated in an oven at 60°C overnight. All scintillation vials containing

213

2 mL of either carcasses, organs or tissues were bleached with 200 µL of H202 and incubated for 2

214

hours in the oven at 60°C.

215 216

2.8.2 Immunization studies

217

Five female C57BL/6 mice (6-10 week-old) per group were injected i.m. (50 μL) into the right

218

quadriceps. Mice received 3 immunizations at 2 week intervals (days 0, 14 and 28) and were

219

terminated 3 weeks after the last immunization (on day 49). For the quantification of serum 9 ACS Paragon Plus Environment

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immunoglobulins, 50 µL of blood was collected via tail-bleed on days 7, 21 and 49. Blood was collected

221

in a heparinised Eppendorf (1% w/v heparin) and centrifuged for 10 minutes at 10,000 x g (Mini

222

centrifuge Mikro200, Hettich (Tuttlingen, Germany) in order to separate blood cells from serum. A

223

standard direct enzyme-linked immunosorbent assay (ELISA) was carried out to detect

224

immunoglobulins IgG1 and IgG2c in serum. Maxisorp 96-well plates flat bottom (Fisher scientific,

225

Loughborough, UK) (high binding and affinity) were coated by passive absorption overnight at 4°C with

226

0.5 µg/mL H56 antigen diluted in carbonate buffer 0.05 N pH 9.6 (100 µL/well). Plates were washed

227

with washing buffer (PBS pH 7.2, 10 mM containing 0.2% Tween 20) and blocked for 1.5 – 2 hours at

228

room temperature with 200 µL/well of PBS (pH 7.2, 10 mM) containing 2% BSA to block any non-

229

specific binding. Samples were added to the plates (serial dilution). Plates were then incubated at

230

room temperature for 2 hours. HRP-conjugated antibody was diluted in 1% BSA in PBS (1:20000 and

231

1:5000 for IgG1 and IgG2c respectively) and added in a volume of 100 µL/well to the washed plates.

232

Plates were incubated for an hour and TMB substrate was added to the plates 100 µL/well. The

233

reaction was stopped by adding 0.2 M sulfuric acid and the absorbance at 450 nm measured (with

234

wavelength correction 570/620 nm). Results were plot as the Log10 of the end point titer giving and

235

optical density value (OD450) of 0.1 or higher.

236 237

Spleens and popliteal lymph nodes from each mouse were isolated and processed on day 49 as

238

described before 35. Cells were counted and diluted in complete RPMI to a final concentration of 2 x

239

106 cells/mL and plated (100 µL) on a Nunclon 96-well round bottom (Fisher scientific, Loughborough,

240

UK). Cells were stimulated with either 100 µL of ConA (5 µg/mL), RPMI media or H56 antigen (5 µg/mL)

241

and incubated at 37°C, 5 % CO2 and 95 % humidity for 72 hours. Supernatants were harvested and

242

stored at -20°C for further processing. The sites of injection (i.e. the right quadriceps) were excised 3

243

weeks after the last immunization (on day 49) and the method from Sharp et al. for the analysis of

244

cytokines at the site of injection was followed 39. Quadriceps muscles were removed from the bone

245

and weighted out individually. Individual muscles were homogenised in 2.5 mL of homogenisation 10 ACS Paragon Plus Environment


Page 12 of 42

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buffer 500 mM NaCl/50 mM Hepes, pH 7.4 containing 0.1% Triton X-100, 0.02% NaN3 and 1% v/v

247

protease inhibitor cocktail. Homogenates were sonicated twice for 15 seconds and centrifuged at

248

3600 rpm 20 minutes at 4°C. Supernatant were removed and stored at -20°C.

249 250

Supernatants from restimulated splenocytes and lymph nodes were analysed using a sandwich ELISA

251

protocol for the production of cytokines IL-17 and IFN-ɣ. Plates were coated with the specific capture

252

antibody diluted in carbonate buffer, overnight at 4°C. Plates were blocked for 1.5 – 2 hours at room

253

temperature with PBS containing 2% milk powder. Samples and standards were diluted in 2% BSA in

254

PBS and incubated at room temperature for 2 hours. Then the biotin conjugate was diluted in 1% BSA

255

in PBS (IL-17 1:2000, IFN-ɣ 1:5000) and 100 µL were added on each well. Plates were incubated for an

256

hour at room temperature followed by incubation with 100 µL/well of HRP-streptavidin in 1% BSA

257

(1:5000). TMB substrate was added to the plates (100 µL/well)And after approximately 15 minutes

258

the reaction was stopped with 0.2 M sulfuric acid. For the quantification of IL-5 cytokine production

259

the manufacturer’s instructions were followed (ELISA IL-5 kit, BD Biosciences). All experiments were

260

carried out in duplicate and absorbance was measured at 450 nm with wavelength correction

261

(570/620 nm).

262 263

2.9 Statistical analysis.

264

All experiments were carried out at least in triplicate unless otherwise stated. Means and standard

265

deviations are plotted on the graphs. Statistical analysis of data was calculated by one-way analysis of

266

variance (ANOVA). Where significant differences are indicated, differences between means were

267

determined by Tukey’s post hoc test.

268 269

3. Results and discussion

270

3.1 Production and optimisation of DDAB:TDB liposomes using microfluidics


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271

The use of microfluidics as a technique for the production of liposomes has already been

272

demonstrated in several studies and process parameters including FRR, TFR and initial lipid

273

concentration all being shown as important considerations

274

parameters were investigated and optimised for the production of DDAB:TDB.

1-3, 35, 40

. Therefore initially, these

275 276

3.1.1 Lipid concentration, flow rate and flow rate ratios.

277

Initial studies considered the impact of initial lipid concentration of the DDAB:TDB liposomes in terms

278

of size, PDI and zeta potential. Previous studies carried out by Davidsen et al. showed that DDAB:TDB

279

immunological responses were optimal when the DDAB:TDB molar ratio was fixed at 8:1 (5:1 weight

280

ratio)22. Therefore, variation of the initial lipid concentration whilst keeping the DDAB:TDB molar ratio

281

constant was examined. Initial liposomes concentrations of 0.3, 1, 2, 3, 4, 6 and 24 mg/mL were

282

formulated at constant microfluidic parameters: TFR of 10 mL/min and FRR of 3:1 (Figure 1). When

283

prepared with low lipid concentrations, the particle sizes were low (approx. 120 nm; Figure 1A).

284

However, at higher concentrations (1 to 24 mg/mL) the particle size was between 250 – 350 nm with

285

no significant differences (Figure 1A). At all concentrations tested, the DDAB:TDB liposomes were

286

more heterogeneous in nature (PDI 0.2 to 0.4; Figure 1B) compared to previously studied liposome

287

formulations (e.g. 1) where PDIs of below 0.2 were achieved. In terms of zeta potential, all formulated

288

liposomes were highly cationic with values between +60 to +75 mV for initial lipid concentrations from

289

1 to 24 mg/mL (Figure 1C). These results show that the DDAB:TDB adjuvants can be formulated at

290

initial lipid concentration between 1 and 24 mg/mL with no significant difference in size, PDI or zeta

291

potential. Previous studies carried out by Joshi et al. have also shown that the initial lipid

292

concentration used for the microfluidic production of liposomes is a critical process parameter; for

293

PC:Chol liposomes it was shown that concentrations above 3 mg/mL gave reproducible

294

physicochemical characteristics of the formed liposomes and at concentrations below this the particle

295

size was increased 3. Forbes et al. also showed that increasing initial lipid concentration from 0.3



Page 14 of 42

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mg/mL to 2 mg/mL decreased the particle size of neutral liposomes (PC:Chol, DMPC:Chol, DSPC:chol

297

and DPPC:chol) and at concentrations above 4 mg/mL the particle size plateaued 1.

298 299

After optimisation of the initial DDAB:TDB concentration for the production of DDAB:TDB, the effect

300

of the TFR and FRR on particle size, PDI and zeta potential was evaluated (Figure 1D and 1E

301

respectively). At all three flow rates tested (5, 10 and 15 mL/min) it can be seen than increasing the

302

FRR (from 1:1 to 5:1) reduces the liposome size and through the combined selection of FRR and TFR

303

liposome sizes of 1000 nm to 160 nm could be prepared (Figure 1D) which, were highly cationic in

304

nature (Figure 1E). However, at lower FRRs, the increased particle size was also paired with high PDI

305

(0.7 to 0.8; Figure 1D). The smallest particle size and PDI combination was achieved at a FRR of 3:1

306

and a TFR of 10 mL/min (approx. 250 nm; 0.3 PDI; Figure 1D).

307 308

The TFR and FRR are important factors to consider when producing liposomes as they impact on the

309

polarity of the organic solvent-aqueous phases when mixing within the micromixer and therefore,

310

influence the physicochemical attributes of the produced liposomes 2, 41. The influence of the FRR has

311

been reported previously in several studies and it is noted that increases in the aqueous phase during

312

the liposome production creates a narrow solvent stream which consequently favours the production

313

of small size particles due to reduced particle fusion 42. Indeed, studies by Kastner et al. 2, Joshi et al.

314

3

315

with neutral or anionic liposomes. Results from another study from Kastner et al. also demonstrated

316

this flow rate/particle size interaction with cationic liposomes containing 1,2-dioleoyl-3-

317

trimethylammonium-propane (DOTAP) 40. Studies carried out with different microfluidic technology

318

also report the effect of the FRR on particle size 40, 43-45. In addition to higher flow rates producing a

319

smaller solvent stream, the lower organic solvent concentration may also reduce particle fusion and

320

subsequently promote the formation of larger particles, 40, 45. Regarding PDI, the high values obtained

, and Forbes et al 1 have shown that increasing the FRR from 1:1 to 5:1 reduced the liposomes size


Page 15 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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for FRR 5:1 may result from the high dilution, lower lipid concentrations, lower diffusion rates and

322

more variable nucleation 40, 46.

323 324

Interestingly, with the manufacture of DDAB:TDB, the speed at which the particles are manufactured

325

through the system was shown to impact on the liposome size contrary to previous studies

326

Generally with microfluidics, the rapid mixing within the microfluidic cartridge results in the organic

327

solvent being diluted very quickly, hindering the formation of large particles

328

DDAB:TDB dissolved in 2-propanol (IPA), high flow rates (> 10 mL/min) combined with higher (>3:1)

329

flow rate ratios may promote better mixing and nanoprecipitation of DDAB and TDB to form

330

liposomes. These results highlight the importance of considering the liposome composition when

331

designing the microfluidic production process. In the case of DDAB:TDB, the particle size of DDAB:TDB

332

liposomes was controlled by both the FRR and TFR. Based on these results a TFR of 10 mL/min was

333

selected for producing liposomes in further studies. This was due to the ability to produce liposomes

334

in different particle size ranges (combined with the lower PDI) through control of the FRR.

1, 46, 47

.

48

. In the case of

335 336

3.1.2 Antigen loading and lipid recovery of DDAB:TDB liposomes.

337

Followed optimisation of the microfluidic method, DDAB:TDB liposomes were produced using the LH

338

as well as microfluidics (FRR 1:1, 3:1 and 5:1 at TFR 10 mL/min) and the particle size, zeta potential,

339

antigen loading and lipid recovery measured (Figure 2). DDAB:TDB produced by the LH were

340

approximately 600 nm in size, with a PDI of 0.3, and a highly cationic surface charge of +80 mV which

341

promotes high (>90%) antigen loading (Figure 1A to C respectively) in line with previously reported

342

studies 13, 22, 49. Addition of 0.1 mg/mL H56 antigen generally results in a small increase in size and PDI,

343

and reduction in zeta potential as would be expected from the addition of an anionic sub-unit antigen

344

to a cationic liposome formulation (Figure 2). Results from DDAB:TDB produced via microfluidics

345

followed a similar trend, with particle sizes tending to increase irrespective of initial particle size, and

346

all formulations showing a high zeta potential and high (> 90%) antigen loading (Figure 2A to C), with 14 ACS Paragon Plus Environment


Page 16 of 42

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no significant difference in antigen loading across the different DDAB:TDB formulations tested (Figure

348

2C). In terms of lipid recovery, across all 4 formulations lipid recovery of both DDAB and TDB was high,

349

with no loss in the microfluidic process, ensuring the 5:1 weight ratio of DDAB to TDB was maintained

350

after production (Figure 2D).

351 352

3.2 Liposome and antigen movement from the injection site to the draining lymphatics.

353

DDAB:TDB liposomes were labelled with 3H-tritium cholesterol and the H56 antigen was labelled with

354

125

355

technologies after intramuscular injection was studied. All four liposomal formulations tested

356

contained the same total lipid concentration (300 µg total lipid and 5 µg H56 per 50 µL dose). The

357

percentage of liposomes (Figure 3A) and antigen (Figure 3B) at the site of injection (right quadriceps)

358

at various time points and the area under the curve (AUC) for each formulation was measured. The

359

method of DDAB:TDB (and the resulting particle size and PDI) does not make a significant difference

360

on the clearance of the liposomes nor the antigen from the injection site, as shown by the similar

361

clearance profiles and AUC, which are not significantly different across the 4 formulations (Figure 3).

362

Drainage from the injection site followed a similar trend for all 4 liposomes formulations, with

363

between 90 and 100% of the dose remaining after 24 hours, dropping to approximately 50% after 8

364

days (Figure 3A). These results for DDAB:TDB produced by LH method (which was used as our control)

365

are in line with previously published data, showing that 1 day post injection, DDAB:TDB liposomes

366

begin to drain steadily from the injection site and on day 8 between 40 - 60% of the initial dose is still

367

detectable there 8. The depot effect and slow clearance from the injection site of DDAB:TDB may be

368

attributed to the cationic nature of the liposomes, which results in the cationic liposomes aggregating

369

with interstitial proteins and becoming trapped at the injection site irrespective of their difference in

370

size. Indeed, previous work investigating the role of particle size using DDAB:TDB liposomes prepared

371

via LH and subsequent sonication to produce small (

Scale-independent microfluidic production of cationic liposomal

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