NANO-01631; No of Pages 10

Nanomedicine: Nanotechnology, Biology, and Medicine

xx (2017) xxx – xxx

nanomedjournal.com

1Q1 Single oral dose of cannabinoid derivate loaded PLGA nanocarriers

2 relieves neuropathic pain for eleven days
E. Berrocoso a, b, c , R. Rey-Brea a , M. Fernández-Arévalo d , J.A. Micó a, b, c ,

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4 Lucía Martín-Banderas d,⁎

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a
5 Neuropsychopharmacology & Psychobiology Research Group, University of Cádiz, Spain
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6 Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Instituto de Salud Carlos III, Madrid, Spain
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Instituto de Investigación e Innovación en Ciencias Biomédicas de Cádiz, INiBICA, Edificio “Andrés Segovia”, Cádiz, Spain

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d
8 Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Sevilla, Sevilla, Spain
9 Received 24 December 2016; accepted 17 July 2017

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10 Abstract

11 Neuropathic pain, resistant to opiates and other drugs, is a chronic/persistent state with a complex treatment and often poor efficacy. In
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this scenario, cannabinoids are increasingly regarded as a genuine alternative. In this paper, and in an experimental animal model of
13 neuropathic pain, we studied the efficacy of three kinds of PLGA nanoparticles containing synthetic cannabinoid CB13: (i) plain
14 nanoparticles (PLGA); (ii) particles coated with PEG chains (PLGA + PEG) and (iii) particles possessing hydrophilic surfaces obtained by
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15 covalently binding PEG chains (PLGA–PEG). The optimized formulation, CB13–PLGA–PEG, showed high drug loading (13%) and small
16 size (b300 nm) with a narrow distribution and controlled surface properties (near-neutral zeta potential and stable PEG corona). Animal
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17 nociceptive behavioral studies were conducted by paw pressure and acetone tests. Versus the free CB13, CB13–PLGA–PEG nanoparticles
18 showed a very noticeable analgesic efficacy with the longest sustained pain-relieving effect, lasting up to eleven days after one oral dose.
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19 © 2017 Published by Elsevier Inc.

20 Key words: PLGA nanoparticles; synthetic cannabinoid; oral administration; neuropathic pain
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22 Neuropathic pain is a chronic/persistent state resulting from has been questioned by many clinicians and indeed, such drugs are 36
23 injury to the nervous system due to trauma, chronic inflammation, not recommended for treating neuropathies. 37
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24 viral infection, or metabolic disturbances, i.e., diabetes. 1 Drug It is evident that new, effective chronic pain management drug 38
25 associations in the routine clinical treatment of neuropathic pain are therapy is required and that there is a drive to find more effective 39
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26 frequently used, including the common use of tricyclic antidepres- treatments. 40

27 sants. Other antidepressants, such as duloxetine or venlafaxine, In recent years there has been growing clinical evidence on 41
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28 anticonvulsants and opioids, are also commonly used. the efficacy of cannabis and synthetic cannabinoid agonists in 42
29 Nevertheless, neuropathic pain treatment is complicated and chronic pain states. Most recently, Science published a special 43
30 often poorly efficacious in the majority of patients. It is resistant to issue dealing with pain research. The section: “The future of pain 44
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31 opiate analgesics 2,3; antidepressants, particularly tricyclic antide- research” 4 indicates that hints are emerging that cannabis could 45
32 pressants, contribute to a poor side-effect profile and limited be one of today's pain management alternatives. 46
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33 patient tolerance 3 due to their anticholinergic, antihistaminergic Cannabinoids are moderately effective in treating neuropathic 47
34 and antiadrenergic properties. The efficacy of non-steroidal pain and new synthetic cannabinoids presenting fewer side effects 48
35 anti-inflammatory drugs (NSAIDs) in treating neuropathic pain have been developed. This includes the potent cannabinoid 49

Financial support information: This work received funding from: Consejería de Innovación, Junta de Andalucía (Spain) (project P09-CTS-5029) and
European funds; Fondo de Investigación Sanitaria (PI13/02659); the Spanish Ministerio de Economía y Competitividad (SAF2015-68647-R), CIBERSAM G18;
Junta de Andalucía (CTS-510, CTS-7748, CTS-480) and 2015 NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation.
⁎Corresponding author at: Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain.
E-mail address: luciamartin@us.es (L. Martín-Banderas).

http://dx.doi.org/10.1016/j.nano.2017.07.010
1549-9634/© 2017 Published by Elsevier Inc.

Please cite this article as: Berrocoso E., et al., Single oral dose of cannabinoid derivate loaded PLGA nanocarriers relieves neuropathic pain for eleven days.
Nanomedicine: NBM 2017;xx:1-10, http://dx.doi.org/10.1016/j.nano.2017.07.010
 2 E. Berrocoso et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2017) xxx–xxx

50 receptor CB1/CB2 agonist 13 (CRA13 or CB13), which reverses CB13 Vehiculization Capabilities 105
51 neuropathic mechanical hyperalgesia. 5 Like other cannabinoid
52 compounds, CB13 is a highly lipophilic drug and belongs to class II Drug content into the nanoparticles was determined by a 106

53 compounds (low solubility and high permeability) of the previous validated and verified HPLC method. 15 Briefly, 5 mg of 107

54 Biopharmaceutics Classification System. In fact, in preclinical lyophilized nanoparticles was accurately weighed. 1 mL of 108

55 studies this drug was orally administered dissolved in a acetonitrile was then added to dissolve the particles. After 109

56 non-aqueous solvent or dispersed in an aqueous phase by the aid this, 10 μL of the previously-filtered solution was injected into the 110
HPLC system for CB13 detection (see Supplementary Materials). 111
57 of dispersing agents such as Cremophor®. 5,6 In addition, due to
CB13 release experiments were performed using the three 112
58 CB13's high lipophilic nature, Trevaskis et al 7 studied the
PLGA-based nanoparticles. 4 mg of nanoparticle samples was 113
59 influence of food on its oral bioavailability. They demonstrated
suspended in 15 mL of a release medium: hydrochloric acid media, 114
60 that the quantity and composition of food can enhance CB13 oral
pH = 2.0 ± 0.1 or phosphate buffered saline, PBS, pH = 7.4 ± 115
61 bioavailability by stimulating lymphatic transport.

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0.1 to simulate gastric and intestinal conditions, respectively (see 116
62 The development of new techniques enabling lipophilic
63 drugs to be administered orally is still a major challenge. In the Supplementary Materials). 117

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64 search for solutions to these problems, nanotechnology-based
In Vivo Studies of CB13-Loaded Nanoparticles in an Animal 118
65 drug delivery systems are promising tools. Moreover, the use of
Neuropathic Pain Model 119
66 nanocarriers in the management of pain is a novel and exciting

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67 area of research, with a great potential for growth and clinical Animals 120
68 benefit. 8 Adult male Harlan Sprague–Dawley rats weighing 250-300 g 121

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69 We have recently developed surface-modified PLGA nano- were provided by the Experimental Unit of the University of Cádiz 122
70 particles and solid lipid nanoparticles intended for oral CB13 (registration number ES110120000210). All the experimental 123

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71 administration. 9,10 In vitro and ex vivo mucoadhesive properties protocols were approved by the Committee for Animal Experimen- 124
72 were enhanced using several mucoadhesive actives such as tation at the University of Cádiz (Spain) and they complied with the 125
73 chitosan or Eudragit®. Nevertheless, in vivo biodistribution International Association for the Study of Pain ethical guidelines. 16
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74 assays revealed that most particles accumulated in the liver and All procedures relating to animal care and use conformed to 127
75 spleen, indicating that nanoparticles did not prevent the European Ethical Standards (86/609-EEC) and Spanish Law (RD 128
76 opsonization process. 9
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1201/2005). All efforts were made to minimize animal suffering. 129
77 In this respect, polyethylenglycol (PEG)-coated nanoparticles
78 appear to be the most promising strategy for several reasons as
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Drugs 130
79 they: (i) promote nanoparticles mucoadhesion; (ii) stabilize the All formulations were administered orally by gavage in a 131
80 nanoparticles in digestive fluids; (iii) avoid plasma protein
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volume of 2 mL/kg. Control animals received DMSO or blank 132

81 adsorption; (iv) minimize the interaction with phagocytic cells; nanoparticles (with no drug) in the same amounts of loaded 133
82 and (v) increase the blood circulating time. 11,12 nanoparticles corresponding to a CB13-equivalent dose of 1.7, 134
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83 The objective of the present work is, therefore, to undertake 3.4 and 6.8 mg/kg, respectively. 135
84 practical research into the potential of PLGA nanoparticles as
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85 oral delivery systems for CB13. For this purpose, free and Neuropathic Pain Model 136
86 nanoparticle-encapsulated CB13 was orally administrated at Chronic constriction injury (CCI) was used as a model of 137
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87 different doses to an animal model of neuropathic pain. neuropathic pain because it induces clinical signs of hypersensitivity 138
88 We prepared three kinds of PLGA nanoparticles that possessed that mimic human conditions of neuropathic origin. CCI was 139
89 either unmodified hydrophobic surfaces or hydrophilic surfaces. produced as previously described 17,18 after anesthetizing the rats with
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90 These were obtained by coating them with PEG chains that were an intraperitoneal injection of 100 mg/kg ketamine and 20 mg/kg 141
91 either adsorbed or covalently attached. 13 xylazine. The left sciatic nerve was exposed at the mid-thigh level 142
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proximal to the sciatic trifurcation, and four chromic gut (4/0) 143
ligatures were tied loosely around the nerve, 1.0 to 1.5 mm apart, 144
92 Methods
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so that the vascular supply was not compromised. Sham operations 145

93 Synthesis of the PLGA-Based Nanoparticles were performed in the same manner but with no nerve ligation. 146
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94 PLGA-based nanoparticles were prepared by a modified Nociceptive Behavioral Assessment 147

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95 nanoprecipitation (NPP) method. 14 Briefly, a weighed amount of
96 PLGA or PLGA–PEG was co-dissolved with Span® 60 in acetone. Paw Pressure Test. Mechanical threshold was determined using 148
97 5 mL of this acetonic solution was then added dropwise under the paw pressure test. 19,20 Briefly, increasing pressure was 149
98 magnetic stirring to 15 mL of a Pluronic® F-68 aqueous solution gradually applied to the dorsal side of the paw using a graded 150
99 (0.5% w/v). The acetone content was evaporated at room motor-driven device (Ugo Basile, Comerio, Italy) with an initial 151
100 temperature (r.t.) for 4 h. Finally, the nanoparticles suspension 30 g of pressure. Two measurements were taken for each paw at 152
101 was collected by ultracentrifugation. To prepare PEG 5-min intervals and the average value was determined, with a 250 g 153
102 coated-PLGA (PLGA + PEG) nanoparticles, PLGA nanoparticles cut-off applied to prevent damage to the paw. Mechanical 154
103 were incubated in a PEG (4.5% w/v) (see Supplementary hypersensitivity is indicated by a reduction in the pressure 155
104 Materials). provoking withdrawal. Nociceptive behavior was assessed in both 156
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t1:1 Table 1
t1:2 Characterization of plain and PEGylated PLGA nanoparticles (mean ± standard deviation (SD); n = 3) (theoretical drug loading (DL) = 16.5% w/w).
t1:3 Formulation D (nm) ± SD PdI ± SD ZP (mV) ± SD EE (%) ± SD DL (%) ± SD
t1:4 PLGA 196.0 ± 12.6 0.188 ± 0.024 −31.4 ± 5.89 73.4 ± 8.0 12.1 ± 0.4
t1:5 PLGA + PEG 654.4 ± 185.5 0.315 ± 0.051 0.45 ± 3.18 79.4 ± 9.0 13.1 ± 0.6
t1:6 PLGA–PEG 207.6 ± 24.5 0.262 ± 0.107 −24.97 ± 4.11 80.1 ± 8.2 13.2 ± 0.5

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162
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160
159
158
157 the ipsi- and contralateral paws. The test was performed by an tion method is based on the drug solubility. It is based on interfacial 207
165 experimenter who was unaware of the treatment condition. deposition of a polymer after displacing a water-miscible 208
semipolar solvent from a lipophilic solution containing CB13 209
166 Acetone Test. To assess the sensitivity to non-nociceptive and PLGA. This method provides high encapsulation efficiency 210
thermal stimuli, a drop of acetone (100 μL) was placed gently

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for drugs presenting low water solubility. 22 211
168 on the plantar surface of the ipsilateral hind paw. Acetone was Table 1 illustrates the size characteristics of the nanoparticles 212

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169 applied alternately five times, with a 5 min delay between each obtained. The three formulations: PLGA; PLGA + PEG; and 213
170 successive application. Responses were monitored for 1 min after PLGA–PEG nanoparticles containing CB13 presented mono- 214
171 acetone application and they were graded on a 4-point scale as disperse profiles and narrow size distributions. 215

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172 described previously 16,21: 0, no response; 1, quick withdrawal, PLGA + PEG nanoparticles presented the largest size, which 216
173 flick or stamp of the paw; 2, prolonged withdrawal or repeated may be attributed to the adsorption of the PEG chains onto the 217

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174 flicking of the paw; 3, repeated flicking of the paw with persistent PLGA surface in the form of one or more layers. 23 This PEG 218
175 licking directed at the ventral side of the paw. The cumulative adsorption could, perhaps, be attributed to van der Waals forces, 219
176 scores were then obtained by summing the four scores for each rat

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hydrogen bonding, apolar interactions, etc., and not by an 220
177 and dividing by 5 (the number of assays). The responses were electrostatic interaction. The other two formulations presented a 221
178 scored by an observer who was unaware of the treatment condition. smaller diameter (≈200 nm). 222
ζ value was found to be lowest (−31.4 ± 5.9 mV) in the case
179 Experimental Protocols
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of plain PLGA nanoparticles due to their carboxyl end groups. 224
PLGA + PEG nanoparticles have shown the highest ζ values 225
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180 Experiment 1: Evaluation of the Effectiveness of PLGA
181 Nanoparticles (Plain) in the CCI Model. On day 7 after (≈+ 0.5 mV), perhaps due to the displacement of the diffuse 226
ionic layer onto the particle surface to greater distances. The 227
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182 surgery, PLGA (1.7-6.8 mg/kg) was orally administered once.

183 Mechanical pain hypersensitivity was evaluated using the paw change in ζ values is a simple means for estimating the extent of 228
surface shielding provided by PEG. These almost neutral ζ 229
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184 pressure test at 0.5, 3, 9 and 24 h after administration. Sensorial

185 threshold was evaluated once a day on days 3 and 5. The same values suggested the highest PEG chain density on particle 230

186 protocol was used for free CB13 (3.4 mg/kg, p.o.). This time surface. 24,25 231
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187 schedule was designed based upon pilot experiment results. Finally, the geometry and surface of the particles and the quality 232
of the PLGA suspensions did not vary significantly when they 233
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188 Experiment 2: Evaluation of the Effectiveness of PLGA + PEG were loaded with CB13. No presence of aggregates or bulky 234

189 Nanoparticles (PEG Adsorbed) in the CCI Model. To study sediments was observed in any of the formulations. Figure 1, A-D) 235
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190 the effect of PLGA + PEG, the same protocol as that used for show SEM images of different PLGA nanoparticles assayed. As it 236

191 PLGA was employed. The selection of this time schedule was can be seen, particles kept their size and spherical morphology. 237
Nevertheless, we observed some capillary bridging between 238
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192 also based upon pilot experiment results.

PLGA + PEG individual particles. It is probably due to an 239
193 Experiment 3: Evaluation of the Effectiveness of PLGA incomplete washing process before collecting nanoparticles by 240
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194 Nanoparticles (PEG Covalently Bind) in the CCI Model. 7 days ultracentrifugation (PEG bridges). 241
195 after surgery, PLGA–PEG (1.7-6.8 mg/kg) was orally administered Encapsulation efficiency (EE %) and drug loading (DL %) were 242
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196 once. Mechanical pain hypersensitivity was evaluated using the paw determined by directly dissolving nanoparticles in acetonitrile 243
197 pressure test at 0.5, 3, 9 and 24 h after administration. Sensorial (Table 1). We found values of around 80% for EE and 13% for DL, 244
198 threshold was also assessed once a day on days 3, 5, 7, 9 and 11. The respectively. This high drug incorporation efficiency may be 245
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199 same protocol was used for free CB13 (3.4 mg/kg, p.o.). This time attributed to the organically soluble nature of CB13 that prevents 246
200 schedule design was based upon pilot experiment results. The partitioning into the aqueous phase, thus increasing drug 247
201 acetone test was also performed 2 days after surgery in both groups. entrapment in nanoparticles during polymer deposition. The high 248
DL (%) values that were found enable the same amount of drug to 249
be delivered with less polymer. The method used for nanoencap- 250
202 Results sulation resulted in a significant enclosure of CB13 and the process 251
was found to be highly reproducible. 252
203 Preparation of the CB13-Loaded PLGA, PLGA + PEG and
As the values between batches were not significantly different 253
204 PLGA–PEG Nanoparticles
(P N 0.05), PEG coating did not influence the encapsulation, 254
205 The nanoparticles containing CB13 were successfully obtained probably due to the process being performed in an aqueous 255
206 by the nanoprecipitation method. The choice of a nanoencapsula- solution where the drug is not soluble. Similar results were 256
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Figure 1. Scanning electron microscopy (SEM) micrographs corresponding to: (A) blank plain-PLGA nanoparticles (from Ref. 10); (B) CB13 plain-PLGA
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nanoparticles (from Ref. 15; (C) blank PLGA–PEG nanoparticles; and (D) blank PLGA + PEG nanoparticles.
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257 obtained for PLGA–PEG. Indeed, the hydrophilic character of PLGA–PEG and PLGA + PEG nanoparticles, where the initial 288
258 PEG kept it oriented toward the aqueous phase, while the 24 h period released 43% and 23% of drug followed by a sustained 289
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259 hydrophobic core of PLGA can entrap the hydrophobic drugs. release to a total of 90% and 82%, respectively, over 15 days 290
260 Since EE% of three formulations was similar, we investigated a of assay. 291
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261 possible interaction between drug to PLGA or PEG or both. For this CB13 release from PLGA nanoparticles was the slowest. In 292
262 purpose a spectroscopy analysis using Fourier transform infrared this case drug release was progressive, to a total of 58% after 293
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263 (FT-IR) was carried out (see Supplementary Materials). Results 15 days. It is possible that CB13 strongly interacts with the 294
264 revealed no significant differences in the frequencies of the bands in PLGA matrix, thus retarding the release capability, and that the 295
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265 the three nanoparticle formulations compared to the single PEG can increase the wettability of the polymeric surface and 296
266 substances (CB13 and the components of the polymeric matrix) matrix, contributing to the increase in drug release. 297
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267 that could indicate the presence of any kind of interaction such as These differences may be attributed to the hydrophilic 298
268 hydrogen bond, for example. In addition, CB13 cannot act as character of the PEGylated nanoparticles thanks to the existence 299
269 hydrogen donor in order to form hydrogen bond with PLGA or PEG. of the PEG chains on the nanoparticle surface which accelerated 300
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270 The in vitro release of CB13 from nanoparticles was the degradation (hydrolysis) rate by improving buffer penetration 301
271 evaluated. CB13 release profiles were obtained by graphing into the nanoparticle matrix. 10,25-28 Thus, surface modification 302
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272 the cumulative percentage of the drug released with respect to the of PLGA nanoparticles with PEG could be advantageously used 303
273 amount of CB13 encapsulated as a function of time. The in order to modulate drug release (nanoparticle erosion) kinetics. 304
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274 experiment was performed over 15 days.

In Vivo Studies of CB13 Loaded PLGA Nanocarriers in an 305
275 Figure 2 collects the in vitro CB13 release profiles at pH 2.0
Animal Neuropathic Pain Model 306
276 (Figure 2, A) and at pH 7.4 (Figure 2, B) from: PLGA,
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277 PLGA + PEG and PLGA–PEG nanoparticles. Independently of Results for in vivo assays are collected in Figures 3-6. In all cases 307
278 the release medium pH, a similar release profile can be observed. asterisks indicate a significant difference compared to saline 308
279 Remarkably, at pH 2.0 the drug release kinetics can be considered (*P b 0.05 vs. free CB13 control group, #P b 0.05 vs. PLGA + 309
280 slower. Drug release was markedly inhibited in acidic environ- PEG control group, &P b 0.05 vs. free CB13 (3.4 mg/kg)). (0 h: 310
281 ments, e.g., b 10% drug release after 2 h. The lowest release profile pre-drug administration) (see Supplementary Materials). 311
282 was obtained for plain PLGA nanoparticles. The presence of PEG
283 chains determined a faster drug release. For PLGA + PEG and Experiment 1: CB13-Loaded PLGA Nanoparticles 312
284 PLGA–PEG ≈ 6% drug release was obtained after 30 min. The effect of three doses of CB13-loaded PLGA nanoparticles 313
285 At pH 7.4 CB13 release from both PLGA–PEG and (1.7, 3.4 and 6.8 mg/kg CB13) was explored in the paw pressure 314
286 PLGA + PEG nanoparticles was faster than from PLGA nano- test in the ipsi- and contralateral hindpaw. This effect was 315
287 particles. A biphasic release pattern of CB13 was observed from compared with those from free CB13 (3.4 mg/kg) (Figure 3). 316
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Experiment 2: CB13-Loaded PLGA + PEG Nanoparticles 344

The effect of three different doses of CB13-loaded PLGA + PEG 345
nanoparticles (1.7, 3.4 and 6.8 mg/kg CB13) was explored in the 346
paw pressure test in the ipsi- and contralateral hind paw over 347
time (Figure 3, C and D). This effect was compared with those from 348
free CB13 (3.4 mg/kg) (Figure 4). We first assessed the effect in the 349
ipsilateral hind paw. Free CB13 showed a similar effect to that 350
previously described (Figure 4, A). PLGA + PEG nanoparticles at 351
3.4 mg/kg CB13 significantly increased the pain threshold 3 h after 352
administration (Figure 4, B). The significant analgesic effect was 353
maintained for 5 days. At 6.8 mg/kg CB13 PLGA + PEG 354
nanoparticles showed a significant analgesic effect from 9 h and 355

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continued for 5 days. The analgesic profile effect of PLGA + PEG 356
nanoparticles at 3.4 and 6.8 mg/kg CB13 was very similar. The peak 357

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effect was found between 9 h and 3 days. No significant analgesic 358
effect was found for the PLGA + PEG nanoparticles at a dose of 359
1.7 mg/kg CB13 (Figure 4, B). 360

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Free CB13 showed an effect similar to that previously 361
described. Its effect was superior to all doses of PLGA + PEG 362

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nanoparticles 0.5 and 3 h after administration. Free CB13 also 363
showed a higher analgesic effect than 1.7 mg/kg of PLGA + PEG 364

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nanoparticles after 9 h (Figure 4, C). PLGA + PEG nanoparticles 365
(6.8 mg/kg CB13) showed a significant antinociceptive effect 366
compared with free CB13 after 9 h. As before, PLGA + PEG
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nanoparticles (3.4 and 6.8 mg/kg CB13) were more effective at 368
reducing the pain threshold than free CB13 24 h, and 3 and 5 days 369
after administration (Figure 4, C). 370
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Figure 2. Cumulative release in vitro of CB13 from plain PLGA nanoparticles This indicates that CB13-loaded PLGA + PEG nanoparticles 371
(▲); PLGA–PEG (♦) and PLGA + PEG (■) nanoparticles (n = 3; error bar: SD).
have a mechanical antihypersensitivity effect that lasts for up to 372
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5 days. 373
No effect with regard to the contralateral hind paw was found 374
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5 in any group (Figure 4, D-F). 375

317 In agreement with previous data, free CB13 showed a
318 significant analgesic effect in comparison with its control group
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319 at 0.5, 3 and 9 h in the ipsilateral hind paw (Figure 3, A). The peak Experiment 3: CB13-Loaded PLGA–PEG Nanoparticles 376
320 effect was found at 3 h after drug administration. The effect of three doses of CB13-loaded PLGA–PEG 377
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321 The three doses of PLGA nanoparticles explored showed a nanoparticles (1.7, 3.4 and 6.8 mg/kg CB13) was explored in the 378
322 significant and dose-dependent effect at 0.5, 3, 9 and 24 h paw pressure test in the ipsi- and contralateral hind paw over time. 379
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323 compared with its respective control group in the ipsilateral hind This effect was compared with those from free CB13 (3.4 mg/kg) 380
324 paw (Figure 3, B). The highest effects were found at 9 and 24 h. (Figure 5). Free CB13 showed a similar effect to that previously 381
325 This analgesic effect was kept up to 3 days after drug found in the ipsilateral hind paw (Figure 5, A). PLGA–PEG 382
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326 administration (Figure 3, B). nanoparticles (1.7, 3.4 and 6.8 mg/kg CB13) showed a dose- 383
327 In the comparison of free CB13 versus CB13-loaded PLGA dependent analgesic effect over time when exploring the ipsilateral 384
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328 nanoparticles in the effect on the ipsilateral hind paw (Figure 3, C), hind paw (Figure 5, B). PLGA–PEG nanoparticles (1.7 mg/kg 385
329 we found that free CB13 had a superior analgesic effect at the CB13) showed a significant analgesic effect 9 and 24 h as well as at 386
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330 beginning of the treatment (0.5 and 3 h compared with PLGA days 5 and 9 after administration. 3.4 mg/kg CB13 PLGA–PEG 387
331 nanoparticles (1.7, 3.4 and 6.8 mg/kg CB13). However, CB13– nanoparticles showed a significant effect from 3 h until 9 days. The 388
332 PLGA nanoparticles showed a higher analgesic effect at later time highest PLGA–PEG nanoparticles dose (6.8 mg/kg CB13) 389
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333 points. That is, at 9 h and 24 h and at 1–5 days CB13–PLGA showed an analgesic effect from 0.5 up to 11 days. The greatest 390
334 nanoparticles 3.4 and 6.8 mg/kg CB13 had a significant effect effects were found between 24 h and 3 days (Figure 5, B). 391
335 compared with free CB13. CB13–PLGA nanoparticles 1.7 mg/kg Free CB13 showed an effect similar to that previously found. It 392
336 also showed a higher analgesic effect versus free CB13 at 1–5 days. displayed a superior analgesic effect to PLGA–PEG nanoparticles 393
337 Therefore, free CB13 is effective for the first 9 h and the at the beginning (0.5 h for all the doses of PLGA–PEG 394
338 CB13-loaded PLGA nanoparticles are effective for 3 days when nanoparticles and after 3 h for PLGA–PEG nanoparticles 1.7 395
339 exploring the ipsilateral paw. and 3.4 mg/kg CB13) (Figure 5, C). Free CB13 also had a 396
340 No significant effect was seen in any experimental group significant effect versus PLGA–PEG nanoparticles (1.7 mg/kg 397
341 when exploring the sensorial sensitivity of the contralateral paw CB13) after 9 h. After this point, the profile was the contrary. 398
342 (Figure 3, D-F) suggesting that the analgesic effect of CB13-loaded PLGA–PEG nanoparticles (1.7 mg/kg CB13) had a significant 399
343 PLGA nanoparticles and free CB13 is restricted to neuropathy. effect compared with free CB13 after 3 and 5 days. Similarly, the 400
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Figure 3. Effects in the paw pressure test in the CCI model in rat of CB13–PLGA nanoparticles and free CB13 (p.o.). (A) Free CB13 (3.4 mg/kg) vs. control
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group; (B) CB13–PLGA nanoparticles (1.7, 3.4 and 6.8 mg/kg) vs. control group and; (C) free CB13 (3.4 mg/kg) vs. CB13–PLGA nanoparticles (1.7, 3.4 and
6.8 mg/kg) in the ipsilateral hind paw (n = 8–10). (D) Free CB13 (3.4 mg/kg) vs. control group; (E) CB13–PLGA nanoparticles (1.7, 3.4 and 6.8 mg/kg) vs.
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control group and; (F) free CB13 (3.4 mg/kg) vs. CB13–PLGA nanoparticles (1.7, 3.4 and 6.8 mg/kg) in the contralateral hind paw (n = 8–10).
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401 highest doses of PLGA–PEG nanoparticles (3.4 and 6.8 mg/kg ticles have a consistent analgesic effect for up to 11 days. 428
402 CB13) showed a superior effect 24 h–7 days after administration. Furthermore, PLGA–PEG nanoparticles are also effective in 429
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403 The 6.8 mg/kg dose of CB13 PLGA–PEG nanoparticles was thermal hypersensitivity in the neuropathic animal model studied. 430
404 significantly more effective than free CB13 9-11 days after oral Finally, the highest dose of PLGA–PEG nanoparticles evaluated 431
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405 administration (Figure 5, C). (6.8 mg/kg CB13) seems to increase the pain threshold in the 432
406 When exploring the effect of these CB13-nanosystems in the non-injured (contralateral) paw. 433
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407 contralateral hind paw, we found a significant effect of PLGA–

408 PEG nanoparticles (3.4 mg/kg nanoparticles) 24 h after
409 administration versus control group and versus free CB13 434
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Discussion
410 (Figure 5, D and F). Similarly, the highest dose of PLGA–PEG
411 nanoparticles (6.8 mg/kg CB13) had a significant analgesic At this point, it can be concluded that neuropathic pain relief 435
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412 effect from 3 h to 5 days versus control group (Figure 5, D). with CB13 can be clearly modulated using PLGA nanoformula- 436
413 Furthermore, 6.8 mg/kg of CB13 PLGA–PEG nanoparticles tions. It is quite noticeable that both free CB13 and the three 437
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414 displayed a significant effect versus free CB13 24 h and nanosystems assayed showed the same analgesic potency while 438
415 3–7 days after administration (Figure 5, F). therapeutic response exists. The main difference lies in the fact that 439
416 Furthermore, we explored the effect of PLGA–PEG nanopar- CB13-loaded nanoparticles with an optimal design maintain the 440
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417 ticles (1.7, 3.4 and 6.8 mg/kg CB13) in the acetone test (ipsilateral analgesic effect for up to 11 days, while free CB13 exerts pain 441
418 hind paw) 2 days after oral administration (Figure 6). As before, this control for 9 h. It could, therefore, be expected that the longest 442
419 effect was compared with that from free nanoparticles (3.4 mg/kg). analgesic effect (up to 11 days) of CB13–PLGA–PEG could make 443
420 The highest doses of PLGA–PEG nanoparticles (3.4 and 6.8 mg/kg this formulation a good candidate for chronic pain management. 444
421 CB13) showed a clear reduction in the acetone score versus control In the present work, we have used PEG for nanoparticle surface 445
422 group and versus free CB13. Compared with its corresponding modification. Versus the free drug, the huge difference observed 446
423 control group, no effect was found in the dose of free CB13 for CB13–PLGA–PEG on the behavioral and pharmacological 447
424 (3.4 mg/kg) explored. effect of CB13 suggests an increased oral bioavailability. The 448
425 Overall, CB13-loaded PLGA–PEG nanoparticles show the enhanced access to the intestinal lymphatics after oral administra- 449
426 longest mechanical antihypersensitivity effect of the three prepa- tion, as well as the capacity of PLGA matrices to control drug 450
427 rations of nanoparticles evaluated. That is, PLGA–PEG nanopar- delivery, may explain this extremely long therapeutic response. 451
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Figure 4. Effects in the paw pressure test in the CCI model in rat of PLGA + PEG nanoparticles and free CB13 (p.o.). (A) Free CB13 (3.4 mg/kg) vs. control
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group; (B) CB13–PLGA + PEG nanoparticles (1.7, 3.4 and 6.8 mg/kg) vs. control group and; (C) free CB13 (3.4 mg/kg, p.o.) vs. CB13–PLGA + PEG
nanoparticles (1.7, 3.4 and 6.8 mg/kg) in the ipsilateral hind paw (n = 7–4). (D) Free CB13 (3.4 mg/kg) vs. control group; (E) CB13–PLGA + PEG
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nanoparticles (1.7, 3.4 and 6.8 mg/kg) vs. control group and; (F) free CB13 (3.4 mg/kg) vs. CB13–PLGA + PEG nanoparticles (1.7, 3.4 and 6.8 mg/kg) in the
contralateral hind paw (n = 7–4).
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452 It is known that CB13 physicochemical profile and lipoprotein the penetration of hydrophobic drugs and promoting intestinal 477
453 affinity (80% of CB13 plasma protein binding is due to association lymphatic transport after oral administration. 30-32 478
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454 with plasma lipoproteins) promote lymphatic transport. Accessing Nonetheless, other factors concerning nanoparticles must also be 479
455 lymphatics, first-pass CB13 metabolism is circumvented and its taken into account. The particles' size and surface properties suggest 480
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456 oral bioavailability substantially enhanced. 7 that a large proportion of an absorbed dose might be expected to 481
457 A variety of nanoparticle delivery systems targeting intestinal drain into intestinal lymphatic capillaries. 33 Recent literature 33-35 482
458 lymphatic include various fatty acid mono-, di- and triglycerides in points out the main factors for the passive lymphatic targeting of 483
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459 their composition in order to promote association with lymph nanoparticles: biocompatible and biodegradable components, 484
460 lipoproteins. Recently, Attili-Qadr et al 29 reported a significantly carrier and drug stability, zeta potential and hydrophobicity. For 485
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461 enhanced effect of orally-administered docetaxel when this latter is targeting lymphatic vessels, size and hydrophobicity seem to be the 486
462 contained within nanocapsules containing a mixture of glyceryl most important nanocarrier design criteria. 487
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463 tributyrate, oleoyl polyoxylglycerides and PLGA. In this case, the In the case of anionic plain CB13–PLGA, it is expected that 488
464 authors hypothesized that nanocapsules were transported into the nanoparticles will be partially degraded by gastric pH 11 and that they 489
465 intestinal lymphatics after receiving a surface coating of will encounter high electrostatic repulsive forces from the 490
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466 apoproteins and phospholipids during their passage across the negatively-charged intestinal mucus, leading to faster clearance. 36-38 491
467 enterocytes (in effect, the nanocapsules became “lipoproteinated”). A sufficient amount of nanoparticles was, however, viable as the 492
468 The PLGA nanoparticles assayed in the present work did not therapeutic response increased from 9 h to 3 days, as compared to 493
469 include any kind of fatty acid in their composition. We suggest that, free CB13. 494
470 due to its affinity to lipoproteins, the CB13 being released from PLGA + PEG neutral nanoparticles are expected to show a 495
471 nanoparticles from the start can itself “lipoproteinate” the high PEG density on the nanoparticles' surface, protecting them 496
472 nanosystems. It is also important to notice that PLGA nanoparticles from gastric pH and enzymes. When compared to plain 497
473 assayed contain a significant amount of Span®60 (33% w/w related nanoparticles, the presence of a dense PEG coating was translated 498
474 to PLGA). The presence of this non-ionic lipophilic surfactant can into a threefold increase in diameter. This increase would, 499
475 also provide a suitable micro-environment for improving oral however, be less evident through the GI tract due to a partial loss 500
476 bioavailability by reducing interfacial surface tension, enhancing of PEG chains. A higher mucus penetration of these nanoparticles 501
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Figure 5. Effects in the paw pressure test in the CCI model in rat of CB13–PLGA–PEG nanoparticles and free CB13 (p.o.). (A) Free CB13 (3.4 mg/kg) vs. control
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group; (B) CB13–PLGA–PEG nanoparticles (1.7, 3.4 and 6.8 mg/kg) vs. control group and; (C) free CB13 (3.4 mg/kg) compared with CB13–PLGA–PEG
nanoparticles (1.7, 3.4 and 6.8 mg/kg) in the ipsilateral hind paw (n = 8–10). (D) Free CB13 (3.4 mg/kg) vs. control group; (E) CB13–PLGA–PEG nanoparticles
(1.7, 3.4 and 6.8 mg/kg) vs. control group and; (F) free CB13 (3.4 mg/kg) compared with CB13–PLGA–PEG nanoparticles (1.7, 3.4 and 6.8 mg/kg) in the
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contralateral hind paw (n = 8–10).

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blood levels to free CB13; (ii) bigger particle size than plain or 509
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PLGA–PEG nanoparticles (600 nm versus 200 nm); (iii) 510

near-neutral ζ-potential values; (iv) a possible PEG loss preventing 511
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nanoparticles diffusion across mucus and then; (v) a higher or 512

faster nanoparticle clearance. 513
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Finally, moderated anionic PLGA–PEG nanoparticles 514

showed an analgesic effect that lasted for as long as 11 days. 515
Difference in activity of PLGA + PEG and PLGA–PEG 516
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nanoparticles can be explained by the PEGylation procedure. For 517

PLGA + PEG nanoparticles, PEG chains are expected to be 518
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non-strongly anchored on the surface of nanoparticles. This way, it 519

is more than likely that PEG chains go away from nanoparticles 520
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surface in the GI tract. Once in the blood stream, a progressive PEG 521
loss can be followed. This can be translated into a higher 522
opsonization process and a fast clearance from the body. In short, 523
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Figure 6. Effect of CB13–PLGA–PEG nanoparticles (1.7, 3.4 and 6.8 mg/kg,

PLGA–PEG nanoparticles with stable PEG chains anchored onto 524
p.o.) and free CB13 (3.4 mg/kg, p.o.) in the ipsilateral hind paw in the acetone test
in the CCI model in rat. Results are expressed as the mean ± S.D. (n = 7–9). the surface of the nanoparticles presented a half-life higher than 525
PLGA + PEG nanoparticles. 526
As explained in Trevaskis et al, 33 nanoparticle size and 527
502 is expected, followed by a smoother passage through enterocytes surface properties suggest that a large proportion of an absorbed 528
503 and finally a facilitated access to intestinal lymphatics, which dose might be expected to drain into intestinal lymphatic 529
504 would promote CB13 activity for 5 days. capillaries. Trevaskis et al 33 state that: “In general, few studies 530
505 Curiously, CB13–PLGA + PEG (1.7 mg/kg) nanoparticles report nanoparticle bioavailability using detailed pharmacoki- 531
506 did not show a significant analgesic effect at any time. This netic analyses of exposure after oral administration and the 532
507 could be due to a combination of circumstances: (i) a small CB13 extent of lymphatic transport of nanoparticles has rarely been 533
508 amount released from nanoparticles, not enough to achieve similar quantified directly. Whether sufficient quantities of nanoparticles 534
 E. Berrocoso et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2017) xxx–xxx 9

535 are absorbed to deliver a typical therapeutic load is therefore less 3. Micó JA, Ardid D, Berrocoso E, Eschalier A. Antidepressants and pain. 583
536 clear”. Moreover, Lammers et al 39 suggest that, beyond targeting Trends Pharmacol Sci 2006;27:348-54. 584
4. Stern P, Roberts L. The future of pain. Science 2016;354(63112):564-5. 585
537 and beyond numbers, nanomedicine works and what counts is
5. Dziadulewicz EK, Bevan SJ, Brain CT, Coote PR, Culshaw AJ, Davis AJ, 586
538 patient benefit. et al. Naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone: a potent, 587
539 In this sense, we have focused our investigation on the orally bioavailable human CB1/CB2 dual agonist with antihyperalgesic 588
540 therapeutic effect of PLGA nanosystems in pain relief manage- properties and restricted central nervous system penetration. J Med Chem 589
541 ment using an animal model. Results clearly showed an 2007;50:3851. 590
542 ultra-long analgesic effect by means of using oral CB13– 6. Gardin A, Kucher K, Kiese B, Appel-Dingemanse S. Cannabinoid 591
receptor agonist 13, a novel cannabinoid agonist: first in human 592
543 PLGA–PEG nanoparticles.
pharmacokinetics and safety. Drug Metab Dispos 2009;37:827. 593
7. Trevaskis NL, Shackleford DM, Charman WN, Edwards GA, Gardin A, 594
Appel-Dingemanse S, et al. Intestinal lymphatic transport enhances the 595
544 Conclusions post-prandial oral bioavailability of a novel cannabinoid receptor agonist 596

F
via avoidance of first-pass metabolism. Pharm Res 2009;26(6):1486-95. 597
545 We have developed biodegradable nanosystems with different 8. Hua S, Wu SY. The use of lipid-based nanocarriers for targeted pain 598

O
546 surface properties varying PEG surface density and anchoring. The therapies. Front Pharmacol 2013;4:1-7 [article 143]. 599
547 three examples of PLGA nanosystems presented here performed 9. Durán-Lobato M, Martin-Banderas L, Gonçalves LM, Fernández-Arévalo 600
548 the following task: sustained and prolonged neuropathic pain relief M, Almeida AJ. Comparative study of chitosan- and PEG-coated lipid 601

O
and polymeric nanoparticles as oral delivery systems for cannabinoids. 602
549 after just one oral dose of PLGA nanoparticles containing CB13.
J Nanopart Res 2015, http://dx.doi.org/10.1007/s11051-015-2875-y. 603
550 CB13–PLGA–PEG nanoparticles presenting small size, 10. Durán-Lobato M, Muñoz-Rubio I, Holgado MA, Álvarez-Fuentes J, 604

R
551 near-neutral ζ-potential values and stable PEG coating exert an Fernández-Arévalo M, Martín-Banderas L. Enhanced cellular uptake and 605
552 analgesic effect for 11 days after administering just one oral biodistribution of a synthetic cannabinoid loaded in surface-modified 606
607

P
553 dose. This noticeable difference in therapeutic effect duration PLGA nanoparticles. J Biomed Nanotechnol 2014;10(6):1068-79.
554 suggests an enhanced passive lymphatic targeting followed by 11. Tobío M, Sánchez A, Vila A, Soriano I, Evora C, Vila-Jato JL, et al. The 608
role of PEG on the stability in digestive fluids and in vivo fate of PEG-PLA 609
555 systemic drug delivery. Therapeutic effect was clearly
nanoparticles following oral administration. Colloids Surf B Biointerfaces
D 610
556 dose-dependent after orally administering nanoparticles. Fur- 2000;18:315-23. 611
557 thermore, CB13–PLGA–PEG nanoparticles presented a delayed 12. Owens DE, Peppas NA. Opsonization, biodistribution, and pharmaco- 612
558 analgesic effect of up to 0.5 h, 3 h or 9 h as a function of CB13 kinetics of polymeric nanoparticles. Int J Pharm 2006;307:93-102. 613
E
559 doses (6.8, 3.4 or 1.7 mg/kg, respectively). 13.. Martín-Banderas L.; Fernández-Arévalo M., Berrocoso E.; Micó J.A. 614
560 The overall results may advocate the feasibility of reducing Method for a pharmaceutical composition of polymeric nanoparticles for 615
T

561 the dose and suggest that CB13–PLGA–PEG nanoparticles treating neuropathic pain caused by periphereal nerve compression. 616
WO2016128591, 2016 617
562 may be an exciting new therapeutic option for the treatment of
C

14. Fessi H, Puisieux F, Devissaguet JPh, Ammoury N, Benita S. 618

563 neuropathic pain. Nanocapsule formation by interfacial polymer deposition following 619
564 Moreover, these results can open up a new perspective for the solvent displacement. Int J Pharm 1989;55(1):R1-4. 620
E

565 future of therapeutic cannabinoid uses, not only with regard to its 15. Álvarez-Fuentes J, Martín-Banderas L, Muñoz-Rubio I, Holgado MA, 621
566 use in pain management, but also with regard to its wide Fernández-Arévalo M. Development and validation of an RP-HPLC 622
R

567 therapeutic spectra. method for CB13 evaluation in several PLGA nanoparticle systems. 623
ScientificWorldJournal 2012737526. 624
16. Flatters SJ, Bennett GJ. Ethosuximide reverses paclitaxel- and vincristine- 625
R

568
Q7 Acknowledgments induced painful peripheral neuropathy. Pain 2004;109:150-61. 626
17. Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that 627
569 We are very grateful to Mr. José Antonio García Partida for produces disorders of pain sensation like those seen in man. Pain 628
O

570 his excellent technical assistance. We also thank the Universidad 1988;33(1):87-107. 629
571 de Sevilla's CITIUS Microscopy and Biology Services for their 18. Berrocoso E, De Benito MD, Mico JA. Role of serotonin 5-HT1A and 630
631
C

opioid receptors in the antiallodynic effect of tramadol in the chronic

572 technical assistance
constriction injury model of neuropathic pain in rats. Psychopharmacology 632
(Berl) 2007;193:97-105, http://dx.doi.org/10.1007/s00213-007-0761-8. 633
N

19. Randall LO, Selitto JJ. A method for measurement of analgesic activity 634
573 Appendix A. Supplementary data on inflamed tissue. Arch Int Pharmacodyn Ther 1957;111:409-419. 635
20. Alba-Delgado C, Borges G, Sánchez-Blázquez P, Ortega JE, Horrillo I, 636
U

574 Supplementary data to this article can be found online at Mico JA, et al. The function of alpha-2-adrenoceptors in the rat locus 637
575 http://dx.doi.org/10.1016/j.nano.2017.07.010. coeruleus is preserved in the chronic constriction injury model of 638
neuropathic pain. Psychopharmacology (Berl) 2012;221(1):53-65. 639
21. Bravo L, Mico JA, Rey-Brea R, Camarena-Delgado C, Berrocoso E. Effect 640
576 References of DSP4 and desipramine in the sensorial and affective component of 641
neuropathic pain in rats. Prog Neuropsychopharmacol Biol Psychiatry 642
577 1. Dworkin RH, O'Connor AB, Backonja M, Farrar JT, Finnerup NB, 2016;3(70):57-67. 643
578 Jensen TS, et al. Pharmacologic management of neuropathic pain: 22. Martín-Banderas L, Álvarez-Fuentes J, Durán-Lobato M, Prados JC, 644
579 evidence-based recommendations. Pain 2007;132:237-51. Melguizo C, Fernández-Arévalo M, et al. Cannabinoid derivate- 645
580 2. Attal N, Cruccu G, Haanpää M, Hansson P, Jensen TS, Nurmikko T, et al. loaded PLGA nanocarriers for oral administration: formulation, 646
581 EFNS guidelines on pharmacological treatment of neuropathic pain. Eur J characterization, and cytotoxicity studies. Int J Nanomedicine 647
582 Neurol 2006;13:153-1169. 2012;7:5793-806. 648
 10 E. Berrocoso et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2017) xxx–xxx

649 23. Parveen S, Sahoo SK. Long circulating chitosan/PEG blended vivo correlation using Caco-2 cells and a cannulated rat intestinal 675
650 PLGA nanoparticle for tumor drug delivery. Eur J Pharm lymphatic model. Pharm Res 2004;21:2320-6. 676
651 2011;670(2–3):372-83. 32. Karpf DM, Holm R, Garafalo C, Levy E, Jacobsen J, Müllertz A. Effect 677
652 24. Jain RA. The manufacturing techniques of various drug loaded of different surfactants in biorelevant medium on the secretion of a 678
653 biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials lipophilic compound in lipoproteins using Caco-2 cell culture. J Pharm 679
654 2000;21(23):2475-90. Sci 2006;95:45-55. 680
655 25. Khalil NM, Frabel do Nascimento TC, Casa DM, Dalmolin LF, de 33. Trevaskis NL, Kaminskas LM, Porter CJH. From sewer to saviour—- 681
656 Mattos AC, Hoss I, et al. Pharmacokinetics of curcumin-loaded PLGA targeting the lymphatic system to promote drug exposure and activity. 682
657 and PLGA–PEG blend nanoparticles after oral administration in rats. Nat Rev 2015;14:781-803. 683
658 Colloids Surf B Biointerfaces 2013;101:353-60. 34. Singh I, Swami R, Khan W, Sistla R. Lymphatic system: a prospective 684
659 26. Corrigan OI, Li X. Quantifying drug release from PLGA nanoparticu- area for advanced targeting of particulate drug carriers. Expert Opin 685
660 lates Eur. J Pharm Sci 2009;37:477. Drug Deliv 2014;11(2). 686
661 27. Makadia HK, Siegel SJ. Poly lactic-co-glycolic acid (PLGA) as 35. Ghosh S, Roy T. Nanoparticulate drug-delivery systems: lymphatic uptake 687
662 biodegradable controlled drug delivery carrier. Polymers 2011;3:1377-97. and its gastrointestinal applications. J Appl Pharm Sci 2014;4(06):123-30. 688

F
663 28. Martín-Banderas L, Muñoz-Rubio I, Álvarez-Fuentes J, Durán-Lobato M, 36. Xu Q, Ensign LM, Boylan NJ, Schön A, Gong X, Yang JC, et al. Impact 689
664 Arias JL, Holgado MÁ, et al. Engineering of Δ9-tetrahydrocannabinol of surface polyethylene glycol (PEG) density on biodegradable 690

O
665 delivery systems based on surface modified-PLGA nanoplatforms. nanoparticle transport in mucus ex vivo and distribution in vivo. ACS 691
666 Colloids Surf B Biointerfaces 2014;123:114-22. Nano 2015;9(9):9217-27. 692
667 29. Attili-Qadri S, Karra N, Nemirovski A, Schwob O, Talmon Y, Nassar T, 37. Date A, Hanes J, Ensign LM. Nanoparticles for oral delivery: design, 693

O
668 et al. Oral delivery system prolongs blood circulation of docetaxel evaluation and state-of-the-art. J Control Release 2016;240:504-26. 694
669 nanocapsules via lymphatic absorption. Proc Natl Acad Sci 38. Ensign LM, Cone R, Hanes J. Oral drug delivery with polymeric 695
670 2013;110:17498-503. nanoparticles: the gastrointestinal mucus barriers. Adv Drug Deliv Rev 696

R
671 30. Kalepu S, Manthina M, Padavala V. Oral lipid-based drug delivery 2012;64:557-70. 697
672 systems—an overview. Acta Pharm Sin B 2013;3(6):361-72. 39. Lammers T, Kiessling F, Ashford M, Hennink W, Crommelin D, Storm 698
673 699

P
31. Seeballuck F, Lawless E, Ashford MB, O'Driscoll CM. Stimulation of G. Cancer nanomedicine: is targeting our target? Nat Rev Mater
674 triglyceride-rich lipoprotein secretion by polysorbate 80: in vitro and in 201616069, http://dx.doi.org/10.1038/natrevmats.2016.69. 700
701
702 D
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