Download Keywords: lymphatic targeting, nanomedicine, mediastinum

Safe Lipid Nanocapsules-based Gel technology to Target Lymph Nodes and
Combat Mediastinal Metastases from an Orthotopic Non-Small-Cell Lung Cancer
model in SCID-CB17 mice
Nathalie WAUTHOZ 1,2, Guillaume BASTIAT 1,2,*, Elodie MOYSAN 1,2, Anna CIESLAK 1,2,
Kazuya KONDO 3, Marc ZANDECKI 4, Valérie MOAL 5, Marie-Christine ROUSSELET 6, José
HUREAUX 1,7 and Jean-Pierre BENOIT 1,2
Word count for the abstract: 144
Word count for the manuscript (including body text, and figure and table legends):
5073
Number of references: 40
Number of figures: 3
Number of tables: 3
1
Safe Lipid Nanocapsules-based Gel technology to Target Lymph Nodes and
Combat Mediastinal Metastases from an Orthotopic Non-Small-Cell Lung Cancer
model in SCID-CB17 mice
Nathalie WAUTHOZ 1,2, Guillaume BASTIAT 1,2,*, Elodie MOYSAN 1,2, Anna CIESLAK 1,2,
Kazuya KONDO 3, Marc ZANDECKI 4, Valérie MOAL 5, Marie-Christine ROUSSELET 6, José
HUREAUX 1,7 and Jean-Pierre BENOIT 1,2
1
2
LUNAM Université – Micro et Nanomédecines Biomimétiques, F-49933 Angers, France
INSERM – U1066 IBS-CHU, F-49933 Angers, France ; [email protected],
[email protected], [email protected], [email protected], [email protected]
3
Department of Oncological Medical Services, Institute of Health Biosciences, The University of
Tokushima Graduate School, 18-15 Kuramoto-cho 3 Tokushima, 770-8509, Japan ;
[email protected]
4
Hematology Department, Academic Hospital, Angers, F-49933, France; [email protected]
5
Biochemistry Department, Academic Hospital, Angers, F-49933, France; [email protected]
6
Cell and Tissue Pathology Department, Academic Hospital, Angers, F-49933, France;
[email protected]
7
Pneumology Department, Academic Hospital, Angers, F-49933, France ; [email protected]
2
* To whom correspondence should be addressed:
Tel: +33 244688531, Fax: +33 244688546, E-mail: [email protected]
The authors declare that there are no conflicts of interest.
Funding sources: this work has been realized within the research program LYMPHOTARG
financially supported by EuroNanoMed ERA-NET 09 and by the Région Pays de la Loire.
Abstract
The purpose of this study is the assessment of a gel technology based on a lauroyl derivative of
gemcitabine encapsulated in lipid nanocapsules delivered subcutaneously or intravenously after
dilution to (i) target lymph nodes, (ii) induce less systemic toxicities and (iii) combat mediastinal
metastases from an orthotopic model of human squamous non-small-cell lung cancer Ma44-3
cells implanted in severe combined immunodeficiency mice.
The gel technology targeted mainly lymph nodes as revealed by the biodistribution study.
Moreover, the gel technology induced no significant myelosuppression (platelet count) in
comparison with the control saline group, unlike the conventional intravenous gemcitabine
hydrochloride treated group (p < 0.05). Besides, the gel technology delivered subcutaneously
twice a week was able to combat locally mediastinal metastases from the orthotopic lung tumor
and to delay significantly the death (p < 0.05) as the diluted gel technology delivered
intravenously three times a week.
Keywords: lymphatic targeting, nanomedicine, mediastinum, immunomodulation
3
Background
Lung cancers remain the leading cause of cancer-related mortality around the world [1, 2]. Nonsmall-cell lung cancers (NSCLC) represent 85% of all cases of lung cancers and constituted of
lung cancer histology differing from small cell lung cancers, mainly represented by
adenocarcinomas (~50%), squamous cell carcinomas (~20%) and large cell carcinomas (~10%)
[3-5]. Diagnosed NSCLC are present in local, regional and advanced extent for 15%, 22% and
56% of patients, respectively [6]. Regional lymph nodes invasion by metastases from the primary
tumor is a critical parameter affecting directly the prognosis of patients with NSCLC with a fiveyear survival rate estimated at 42% for a stage of N0 (without regional lymph node metastases) to
7% for N3 corresponding to the latest stage of lymph node implication [3]. Invasion of the
mediastinum lymph nodes corresponds to the N2 and N3 staging with a five-year survival rate of
16% and 7%, respectively [3]. Patients at this stage are considered in the advanced stage III
treated mainly by a combination of radiotherapy and chemotherapy with poor improvement in
long-term survival and at the expense of a large raise in Grade 3 toxicities [7]. Platinum–based
doublet-third generation agent chemotherapy is the first-line chemotherapy having proven
improved survival, response rates, and quality of life for advanced NSCLC patients presenting
good performance status. This gold standard is generally based on a platinum compound
(cisplatin or carboplatin) and associated with a third-generation agent such as a taxane (paclitaxel
or docetaxel), gemcitabine, or vinorelbine [5], which present a similar efficacy [8]. According to
pharmacoeconomic data, cisplatin and gemcitabine regimen are associated with the lowest total
treatment-related costs among platinum-based combinations with third-generation cytotoxic
drugs [9]. In addition, because cisplatin administration induces severe toxicities and also because
a therapeutic plateau is reached, a lot of nonplatinum-based regimens are explored with
4
gemcitabine as the most investigated third-generation drug or in platinum-based combination
with targeted agents (e.g., cetuximab, bevacizumab), respectively [9]. Gemcitabine hydrochloride
also called 2’deoxy-2’2’-difluorocytidine hydrochloride, is a potent and specific pyrimidine
nucleoside antimetabolite which is structurally analogous to deoxycytidine [9, 10]. Due to its
hydrophilic nature and low membrane permeability but also its extensive deamination in plasma
and tissues by cytidine deaminase, its plasma elimination half-life (t1/2) after 30 min-perfusion is
extremely short (i.e., 42-94 min) [10], which requires high dose regimen and leads mainly to
dose-limiting myelosuppression in approximately two-thirds of patients [9]. Moreover, different
mechanisms of resistance against gemcitabine were developed by cancer cells what fostered the
emergence of chemically modified gemcitabine [10]. A 4-(N)-lauroyl prodrug of gemcitabine
(Gem-C12) was synthetized by Tokunaga et al to decrease the hydrophilicity responsible of the
poor drug loading in lipophilic nanosystem and to decrease the deamination in blood responsible
of the inactive metabolite formation [10].
In aim to encapsulate Gem-C12, protect the prodrug until the site of action, target the lymph
nodes invaded by metastases and decrease the related systemic toxicities, nanomedicine could be
of interest [11]. Indeed, some examples of nanotechnology in clinics demonstrated these abilities:
liposome-encapsulated doxorubicin (Doxil®) protects patients with ovarian cancer, breast cancer
or Kaposi’s sarcoma from the cardiotoxicity of the unencapsulated drug; paclitaxel-based
albumin nanoparticles (Abraxane®), approved for metastatic breast cancer, have showed an
increased drug uptake in tumor; iron oxide-based nanoparticles (Ferumoxytol®) is able to stage
early lymph node metastasis in patients with prostate and testicular cancers [11].
A nanocarrier system, lipid nanocapsules (LNC), loaded with the lipophilic pro-drug Gem-C12,
has been developed and has demonstrated the ability (i) to form a hydrogel when Gem-C12 is
5
encapsulated in LNC, (ii) to release Gem-C12-based LNC at a rate depending on the Gem-C12
concentration, (iii) to be in vitro injected through 18 or 21G needle, (iv) to form a suspension
after dilution, and (v) to exert an higher in vitro anticancer activity against human NSCLC and
prostatic carcinoma cell lines [12]. The purposes of this study are to evaluate in vivo this LNCbased gel technology in terms of (i) biodistribution and pharmacokinetics, (ii) tolerance, and (iii)
antitumor efficacy in a highly aggressive patient-like lymphogenous metastatic model of human
NSCLC. This lymphogenous metastatic model mimics the spreading of metastases in
mediastinum from the primary tumor implanted in the lung of severe combined
immunodeficiency mice with CB17 genetic background (SCID-CB17) as observed for NSCLC
patients [13].
Methods
Material
Labrafac® WL 1349 (caprylic–capric acid triglycerides) was provided by Gattefossé S.A. (SaintPriest, France). Kolliphor® HS15 (mixture of free polyethyleneglycol 660 and polyethyleneglycol
660 hydroxystearate) was supplied by BASF (Ludwigshafen, Germany). Sodium chloride,
acetone and ethanol were purchased from VWR (Fontenay-sous-bois, France). Span® 80
(sorbitane monooleate), Tween® 80 (polysorbate 80) and 5-aminolevulinic acid (ALA) were
purchased
from
Sigma
(St
Quentin
Fallavier,
France).
1,1'-Dioctadecyl-3,3,3',3'-
tetramethylindodicarbocyanine (DiD) was provided by Life Technologies (Saint-Aubin, France).
Methanol was analytical grade purchased from Fischer Scientific (Loughborough, United
Kingdom). Water was obtained from a MilliQ filtration system (Millipore, Paris, France). 4-(N)lauroyl-gemcitabine (Gem-C12) was synthesized and characterized as described elsewhere [12].
6
Preparation of the formulations
The LNC-based gel technology was prepared following the phase inversion temperature (PIT)
process
[12, 14]. Briefly, Gem-C12 (0.062g) was first solubilized (5 %, ratio Gem-
C12/Labrafac® w/w) in a mix of Labrafac® WL 1349 (1.24g), Span® 80 (0.25g) and acetone
that was evaporated before the addition of Kolliphor ® HS15 (0.967g), NaCl (0.045g) and
water (1.02g). All were then mixed and heated to 75°C under magnetic stirring at 500 rpm
followed by cooling to 45°C (rate of 5°C/min). Three cycles were performed and at the last
cooling phase, a sudden dilution with 2.12g room-temperature water was carried out at 60°C.
LNC loaded with Gem-C12 form spontaneously a hydrogel with a waxy aspect. Gelation process
was considered as achieved after 24h at 4°C. LNC loaded with the prodrug (Gem-C12 LNC) and
used in a liquid form were acquired by the 4-fold dilution of the formulation directly after the
process before the establishment of the hydrogel. Liquid non-loaded LNC were prepared by the
same way without Gem-C12. Fluorescent LNC were obtained by adding the fluorescent probe
(DiD) with the other reagents at a final concentration of 0.1% of Labrafac (w/w). Free Gem-C12
was dissolved in ethanol, Tween® 80 and water (87.6/5.5/6.9 v/v/v) as a micellar system for in
vivo experiments. Commercial gemcitabine hydrochloride used was Gemcitabine Hospira 38
mg/mL diluted at the required concentration with NaCl 0.9%.
Characterization of LNC-based formulations
Hydrodynamic diameter (Z-average), polydispersity index (PdI) and zeta potential of LNC-based
formulations were determined by dynamic light scattering using a Zetasizer® Nano serie DTS
1060 (Malvern Instruments S.A., Worcestershire, UK). LNC-based formulations were diluted
7
1:60 (v/v) in deionised water in order to ensure a convenient scattered intensity on the detector.
Each measurement was done in triplicate at 25°C. The drug loading was determined after dialysis
using an UPLC apparatus with UV spectroscopy detection as previously described [12]. Each
measurement was done in triplicate.
Animals
For pharmacokinetic and biodistribution studies, female nude SWISS mice were purchased from
Harlan (Gannat, France) and for in vivo efficacy and tolerance evaluations, male SCID-CB17
mice were purchased from Charles River (L’Arbresle, France). The animals are housed and
maintained at the University animal facility (SCAHU) in disposable plastic cages with hardwood
chips bedding in an air-conditioned room with a 12-hour-light-12-hour-dark cycle. All the animal
experiments were carried out in accordance with EU Directive 2010/63/EU and with the
agreement of “Comité d’Ethique pour l’Expérimentation Animale des Pays de la Loire”
(authorization CEEA; 2012-37 and 2012-73).
Pharmacokinetic and biodistribution of fluorescent LNC-based formulations in healthy
nude mice
Mice (9 weeks of age, n = 5 for each group) were anesthetized (isoflurane) and either 110 µL of
DiD- Gem-C12 LNC were injected subcutaneously (sc) behind the neck, or 110 µL of DiD LNC
were injected intravenously (iv) into the tail vein with the aim to deliver the same amount of DiD
loaded LNC. After various times, from 1 h to 336 h, the mice were sacrificed, the blood was
removed by cardiac puncture in a venous blood collection tube containing Li-heparin (Tube
8
Micro from SARSTEDT, Marnay, France). The organs were then removed for the biodistribution
study. Plasma from each blood samples was obtained after 10 min of centrifugation at 2,000 g.
The DiD concentrations in plasma (encapsulated inside LNC) along time were determined using
a microplate reader Fluoroscan Ascent® (Labsystems SA, Cergy-Pontoise, France) at excitation
and emission wavelengths of 646 and 678 nm, respectively. The DiD concentrations in plasma
were intrapolated from linear curve between 0.006 and 12 µg/mL (r2 > 0.999). The recovered
DiD concentrations were then normalized in function of the animal weight, assuming blood
represents 7.5% of mouse body weight [15]. Pharmacokinetic data were determined by iv bolus
and extravascular non-compartmental analysis (for iv and sc administration, respectively) of the
recovered systemic DiD concentration versus time using Kinetica 4.1.1 software (Thermo Fisher
Scientific, Villebon sur Yvette, France). The trapezoidal calculation method was used to
determine the area under the curve (AUC) during the whole experimental period (from 1 to 336
h) without extrapolation. The t1/2 were calculated from 1 to 8 h for t1/2 distribution and from 8 to
336 h for t1/2 elimination.
For the biodistribution study, the organs (i.e., kidneys, liver, spleen, lung, heart, stomach, and
intestine), and lymph nodes (i.e., inguinal, axillary, cervical and brachial lymph nodes) were
removed and analyzed by a fluorescence CRI MaestroTM imaging system (Woburn, USA). Semiquantitative data were obtained by setting a time exposition of 10 ms between 630 and 800 nm,
unmixing the generated cube, extracting the background and drawing the regions of interest from
fluorescence images (Figure 1-A). The software Maestro 2.10 (Woburn, USA) was used to
calculate the average signal expressed in photon/cm²/s.
Cell cultures and inoculum preparation for lung implantation
9
Ma44-3 cell line derived from the human squamous NSCLC carcinoma cancer cell line Ma44 by
limit dilution method was kindly provided by Prof. Kondo (Department of Oncological and
Regenerative Surgery, School of Medicine, University of Tokushima, Japan) and was cultured in
RPMI 1640 (Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine
serum (Lonza, Verviers, Belgium), 100 U/mL of penicillin, 100 µg/mL of streptomycin and
0.250 μg/mL of amphotericin B from Sigma (St Quentin Fallavier, France) at 37°C in a
humidified incubator with 5% CO2. For lung implantation, the cells were harvested at 70-80%
confluence using trypsin that was inactivated by the culture medium. Then, the cell suspension
was centrifuged at 1,400 rpm during 5 min and the supernatant was removed and replaced by
RPMI 1640 containing 0.1% of bovine serum albumin fraction V pH 7.0 (PAA GmbH, Pasching,
Austria) which was then mixed to Matrigel® (DB Biosciences, Bedford, MA) to obtain an
inoculum of 2.0 × 106 tumor cells/mL with 400 µg/mL of Matrigel®. The inoculum is kept on ice
during maximum 2h.
Orthotopic intrapulmonary implantation procedure
The orthotopic intrapulmonary implantation procedure was performed as previously reported
[13]. Briefly, the 6 weeks-aged SCID-CB17 mice were maintained in the right lateral decubitus
and anesthetized by isoflurane inhalation. A 1-cm transverse incision was made on the left lateral
skin just below the inferior border of the scapula of the SCID-CB17 mice. The muscles were
separated from the ribs by sharp dissection, and intercostal muscles were visible. 10 μL of cell
suspension (2.0 × 104 cells) were inoculated with a 30-gauge needle to a depth of about 3-5 mm
into the lung through the intercostal muscles and after, the needle was promptly pulled out. Mice
were maintained in the right lateral decubitus position after injection and the skin incision was
10
closed with 3-0 silk (Ethicon, St-Stevens-Woluwe, Belgium). Mice were observed until complete
recovery.
In vivo antitumor efficacy on the lymphogenous metastatic model
Treatments were started 5 days after the intrapulmonary implantation of Ma44-3 cells. Groups
contained 10 SCID-CB17 mice. At day 5, 7 and 9, NaCl 0.9% solution (saline iv), non-loaded
LNC (non-loaded LNC iv), commercial gemcitabine hydrochloride (Gemcitabine iv), Gem-C12
micelles (Gem-C12 iv) and liquid form of Gem-C12-loaded LNC (Gem-C12 LNC iv) were
administered iv by the tail vein (145 µL at each injection). At day 5 and 9, non-loaded LNC (nonloaded LNC sc) and gel form of Gem-C12-loaded LNC (Gem-C12 LNC sc) were administered sc
behind the neck (55 µL at each injection). The total LNC delivered dose of non-loaded LNC was
the same that Gem-C12-loaded LNC for iv or sc. In groups treated by gemcitabine hydrochloride
or Gem-C12 (in LNC or not), the dose delivered in mice was 40 mg (molar equivalent
gemcitabine hydrochloride) per kilogram of body weight per week. Mice were observed each day
and body weight measurements were performed daily during the treatment period and then three
times a week. By applying the criteria for euthanasia of experimental animals, mice were
sacrificed when they lost 20% of body weight and/or associated with a high degree of respiratory
depression.
Visualization of the fluorescent LNC-based formulations in lung and mediastinum in
tumor bearing mice
The visualization of the lung and mediastinal distribution of fluorescent LNC-based formulations
delivered sc or iv after dilution was performed after 4h on tumor grafted SCID-CB17 mice. At
11
day 5 post tumor grafting, a group of 3 mice received an iv injection of DiD LNC and the other
group of 3 mice received a sc injection of DiD- Gem-C12 LNC to deliver the same amount of
DiD LNC. Moreover, both groups received orally 400 mg/kg of ALA in PBS (200 µL). Four
hours after the administrations, the mice were killed and dissected for imaging the lung and the
mediastinum area. Fluorescence imaging was performed using the CRI Maestro system between
500 and 635 nm (for Pp IX for cancer cells visualization) and 630 and 800 nm (for DiD for LNC
visualization) using the automatic exposition.
In vivo tolerance evaluation of the formulations
Mice (7 weeks of age, 5 per group) received 145 µL of an iv injection of saline (saline iv),
gemcitabine hydrochloride (Gemcitabine iv), Gem-C12 micelles (Gem-C12 iv), non-loaded LNC
(non-loaded LNC iv) or liquid form of Gem-C12-loaded LNC (Gem-C12 LNC iv) dispersions at
day 5, 7 and 9. Ten mice (5 per group) received 55 µL of a sc injection of non-loaded LNC (nonloaded LNC sc) or gel form of Gem-C12-loaded LNC (Gem-C12 LNC sc) at day 5 and 9. The
LNC delivered dose of non-loaded LNC was the same that Gem-C12-loaded LNC for iv or sc
administrations. In groups treated by gemcitabine hydrochloride or Gem-C12 (in LNC or in
micelles), the dose delivered in mice was 40 mg (molar equivalent gemcitabine hydrochloride)
per kilogram of body weight per week. Hematology and biochemical assays on blood were done
for each mouse 8 days after the first injection to assess the tolerability of treatments. Blood
sampling was performed by cardiac puncture in deeply anaesthetized mice, the half of blood
sample was placed in a venous blood collection ethylene-diamine-tetraacetic acid tubes (K2E
tube from BD Microtainer, NJ, USA) for hematology studies and the other half in a venous blood
collection tube containing heparin lithium (Tube Micro from SARSTEDT, Marnay, France)
12
which are then centrifuged at 10,000 g for 10 min to remove the plasma and evaluate the blood
biochemical markers. The hematological parameters were determined in the Hematology Ward of
the Academic Hospital of Angers with an XE-2100 hematology analyzer (Sysmex) and plasma
biochemistry analyses were carried out at the Biochemistry Ward of the Academic Hospital of
Angers on an Architect C16000 (Abbott).
Statistical analysis
Survival analyses were carried out by means of Kaplan-Meier curves and the log-rank test.
Statistical comparisons of more than three independent groups of data were analyzed by a nonparametric one-way analysis of variance (Kruskal-Wallis test), followed by Dunn's post-hoc test
for pairwise comparisons.
Results
LNC-based formulations
The physicochemical characteristics of LNC-based formulations are presented in Table 1. With
the addition of Gem-C12 into LNC, a hydrogel was formed and can be injected using a syringe
[12]. Once the gel was 5-fold diluted in water, liquid LNC suspension was obtained (Gem-C12
LNC iv). LNC formulations performed by the PIT method displayed a Z-average range (i.e.,
hydrodynamic diameter) between 53 and 68 nm, depending on the presence of the amphiphilic
Gem-C12 and/or DiD in the LNC surface, and a polydispersity index in all cases less than 0.1,
which means that monomodal and monodispersed distributions were obtained. In all cases, the
zeta potential was slightly negative, from -5 to -10 mV. Similar to many hydrophobic drugs [16,
13
17], Gem-C12 was well-encapsulated in LNC with a high encapsulation efficiency of about
100%.
Biodistribution and pharmacokinetic of LNC-based formulations
LNC loaded with the fluorescent dye DiD were used to evaluate their tissue biodistribution and
pharmacokinetic after iv (suspension of DiD LNC) and sc (hydrogel of DiD- Gem-C12 LNC)
administration in nude SWISS mice. The fluorescent dye DiD was chosen to be encapsulated in
the LNC because (i) it is a near-infrared fluorophore limiting the autofluorescence wavelength
emitted by animals [18-20], (ii) it is weakly fluorescent in aqueous media, but highly fluorescent
in lipophilic vehicle [21], and (iii) there is no dye release (strong fluorescence labeling stability of
the nanocarrier) [22]. Semi-quantitative fluorescence of the organs extracted after 1, 4, 8, 48, 96,
and 336h was reported in Figure 1-C to F and Figure S1 in supplementary information. DiD
LNC iv or DiD- Gem-C12 LNC sc was rapidly distributed in lymph nodes of nude mice. An
immediate accumulation in liver and spleen after 4 and 8h was observed with DiD LNC iv. After
48 h, a decrease of the accumulation was observed for these organs to totally disappear after 336
h (Figure 1-C). DiD-Gem-C12 LNC sc accumulated exclusively in the lymph nodes close to the
injection site (i.e., in axillary, cervical and brachial lymph nodes) in quite same amount for the
first hours (i.e., 1, 4 and 8h) as DiD LNC iv but much higher after few days (i.e., 48, 96 and
336h) (Figure 1-F). Moreover, DiD-Gem-C12 LNC sc presented a much lower plasmatic
exposition than DiD LNC iv (Figure 1-B) resulting in a lower AUC1-336h of 4 µg/mL/h in
comparison with a AUC1-336h of 174 µg/mL/h, respectively. Besides, DiD LNC were more
rapidly cleared from the systemic circulation after iv administration with a t1/2 elimination of 19 h
14
in comparison to 32h found for the sc injection of the hydrogel. Indeed, the latter presented
controlled-release properties [12].
In vivo antitumor efficacy of the LNC-based formulations
The patient-like lymphogenous metastatic Ma44-3 preclinical model was used to evaluate the
efficacy of LNC-based gel technology. Five days post-tumor graft in the left lobe of lung, when
micrometastatic foci were detected in mediastinum (Figure S2-A), a comparative efficacy study
on randomized groups were performed with different treatments and schedules of administration.
The dose of 40 mg (molar equivalent dose of gemcitabine hydrochloride) by kilogram of body
weight per week delivered twice or three times a week by the sc and the iv routes, respectively,
was chosen because no weight loss or death was observed in a group of three mice using GemC12 LNC (gel form) for sc route or Gem-C12 LNC (liquid form) for iv route, respectively.
Higher doses or similar doses with less injection caused drastic weight loss or death of mice (data
not shown). Survival times of experimental animals were plotted on the Kaplan Meier curves as
shown in Figure 2-A. Mice of the saline iv and non-loaded LNC iv or sc groups were
characterized by a very short lifespan after tumor implantation without significant difference (p >
0.05, log-rank test) (Table S1 in supplementary material). In the treated groups by gemcitabine
hydrochloride or Gem-C12, all groups except Gemcitabine iv (i.e., Gem-C12 iv, Gem-C12 LNC
iv and Gem-C12 LNC sc) showed significant difference (p < 0.05, log rank test) in comparison
with the saline iv group (Table S1 in supplementary material). However, only Gem-C12 LNC sc
showed a significant prolonged lifespan (p < 0.05, log rank test) in comparison to its control group
(i.e., non-loaded LNC sc) and only Gem-C12 LNC iv showed a significant prolonged lifespan (p
< 0.05, log rank test) in comparison to Gemcitabine iv group (Table S1 in supplementary
15
material). The weight evolution of the different mice groups remained similar during the
experiment with a short stabilization during the treatment period (Figure 2-B).
Visualization of fluorescent LNC-based formulations lung and mediastinum
The in vivo behavior of LNC-based formulations was then assessed following the iv injection of
DiD LNC and the sc injection of DiD-Gem-C12 LNC in tumor-bearing SCID-CB17 mice to
better understand the result of antitumor efficacy. The fluorescence images obtained 4h after
DiD-loaded LNC formulations injections are presented in Figure 3. DiD LNC are visible in the
entire lung of the three mice after iv injection (Figure 3-B) because at this time, LNC mainly
remained in the blood as confirmed by plasmatic exposition (Figure 1-B) and because lung is a
highly vascularized organ. For DiD-Gem-C12 LNC administered by sc route, only an intense
local accumulation in mediastinal lymph nodes was observed (Figure 3-D). Mediastinal lymph
nodes are known to be close to thymus [23]. Indeed, a negligible plasma exposition for DiDGem-C12 LNC was previously seen in Figure 1-B. However, no accumulation of DiD LNC (after
iv administration) was seen in mediastinal lymph nodes in contrary to the other lymph nodes
during the biodistribution study (Figure 1-E).
Tolerance of the LNC-based gel technology on SCID-CB17 mice
To evaluate if the different treatments used in SCID-CB17 mice induced myelosuppresion, the
complete blood count was performed 8 days after the first injection as in clinic [24] and are
presented in Table 2. A significant decrease of platelet count was only observed with the group
treated by gemcitabine hydrochloride iv in comparison to the saline iv control group (p < 0.05,
Kruskal-Wallis test) and no difference was measured for Gem-C12 non or loaded in LNC and
16
delivered iv or sc in the respective schedule. SCID-CB17 mice are severe combined
immunodeficiency mice characterized by a severe lymphopenia [25], which prevented the
analyze of the compete granulocyte count. Plasma biochemical parameters such as aspartate
aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (PH ALK)
and serum creatinine were evaluated (Table 3). No significant difference was seen between the
different groups in comparison to the control saline iv group except a significant decrease of PH
ALK for Gem-C12 not loaded in LNC but formulated with ethanol as co-solvent and in surfactant
micelles (p < 0.05, Kruskal-Wallis test).
Discussion
Drug delivery to the lymphatic system is attractive to combat metastasis spreading from a
primary tumor by lymphatic system and to modulate immunity. Indeed, the lymphatic system
filters particles (e.g., nanomedicine) or cells (e.g., cancer cells) from interstitial fluid and supports
the activity of the lymphocytes, which furnish immunity, or resistance, to the specific disease
causing agents [26]. In this study, sc administration route and 50-nm size of LNC composing the
hydrogel were chosen because they are the most appropriate for lymphatic targeting [26-30].
Indeed, after sc administration, Ousoren et al showed that nanocarriers whose size was lower
than 0.1 µm have direct access to lymphatic capillaries and that absorption by blood capillaries is
restricted to water and small hydrophilic molecules [30]. The gel form of DiD-Gem-C12 LNC
released progressively 50-nm LNC in the interstitial fluid passing in the lymphatic capillary
vessels that is drained until lymph nodes as observed by the specific accumulation of DiD LNC
in adjacent lymph nodes such as in axillary, brachial, cervical (Figure 1-F) and mediastinum
lymph nodes (Figure 3-B). In the case where DiD LNC were delivered intravenously, LNC are
17
taken up by the mononuclear phagocytic system recognizing adsorption of complement proteins
on their LNC surface as observed by the rapid accumulation in the liver and spleen (Figure 1-C)
and as previously reported by Hirsjarvi et al [18, 31]. DiD LNC were also partly carried up to
lymph nodes as revealed by their accumulation in all studied lymph nodes (Figure 1-E).
However, they were not accumulated in mediastinum lymph nodes, where is drained the
interstitial fluid of lung. Indeed, healthy blood vessels of lung present poorly permeable
properties due to small pores in postcapillary venules (< 6nm) [32]. By consequent,
nanomedicine such as 50 nm-LNC were poorly extravasated in lung parenchyma and then poorly
transported to mediastinum lymph nodes. Then, the LNC-based gel technology, able to target
more specifically the lymph nodes by subcutaneous route and loaded with a pro-drug of
gemcitabine, Gem-C12, was tested on a patient-like lymphogenous metastatic Ma44-3 preclinical
model. There are a very few preclinical models of lymphogenous metastatic NSCLC in the
literature. Ma44-3 preclinical model developed by Ishikura et al [13], was chosen because i) it is
a subpopulation of a human squamous NSCLC grafted in the lung [13], ii) it spreads in the
mediastinum [13, 33], and iii) it causes the death of mice from the mediastinum metastases and
not from the primary tumor [34].
Despite the presence of the primary lung tumor responsible of the cancer cells spreading to
mediastinum, Gem-C12 LNC only localized in mediastinum lymph nodes were able to exert the
same antitumor efficacy than a systemic administration of Gem-C12 (loaded in LNC or micelles)
(Figure 2-A), which exerted a systemic anticancer activity (i.e., on primary lung tumor and
therefore on the spreading of mediastinal metastases). Moreover, this similar anticancer activity
by targeting only lymph nodes was also accompanied by lower systemic side effects. Indeed,
Gem-C12 LNC delivered by iv or sc routes presented no significant depletion in platelet count, a
18
marker of myelosuppression, contrary to conventional gemcitabine hydrochloride iv (Table 2)
and no significant depletion in alkaline phosphatase contrary to Gem-C12 loaded in surfactant
based micelles (Table 3) when compared to the control saline iv group, respectively. By
consequent, the developed LNC-based gel technology is able to exert a similar anticancer activity
when delivered subcutaneously by targeting locally the adjacent lymph nodes without leading to
myelosuppresion as encountered with conventional chemotherapies. Nowadays, nanomedicine
approved on the market present a better therapeutic index mainly by decreasing drastically
toxicities. For example, Doxil® (for US) and Caelyx® (for Europe), that are pegylated liposomal
doxorubicin, and Myocet®, a non-pegylated liposomal doxorubicin, decrease drastically the
cumulative dose-dependent cardiotoxicity but do not improve the survival in comparison to
conventional doxorubicin [35]. The nab-paclitaxel (Abraxane®), albumin-bound paclitaxel,
overcomes the toxicities and complications encountered with the conventional formulation based
on polyethoxylated castor oil (Cremophor® EL) and ethanol, what allows an increase of the
administrable doses to improve slightly survival [36].
Concerning immunomodulation, the preclinical model used in this study, despite a development
of metastases in mediastinum close to those happening in NSCLC patients, was based on severe
immunodeficiency. Indeed, SCID-CB17 mice present a mutation at the protein kinase, DNA
activated, catalytic polypeptide (Prkdcscid) locus responsible to non-mature T and B cells [37];
necessary to assure a successful human tumor cells grafting. Therefore, the immunologic action
of gemcitabine was not taken into account in the in vivo antitumor activity study. Indeed,
gemcitabine was shown to increase the expression of the major histocompatibility complex
(MHC) on malignant cells, enhancing the cross-presentation of tumor antigens to CD8+ T cells,
and selectively kill myeloid-derived suppressor cells, both in vitro and in vivo, thus facilitating T
19
cell-dependent anti-cancer immunity [38]. Good results have been obtained combining
gemcitabine and immunotherapy in murine model of pancreatic carcinoma and in non-resectable
pancreatic patients during phase II clinical trial [39, 40]. So, further investigations are required to
evaluate the impact of this localized treatment in lymph nodes to enhance immunity against
tumor and metastases.
Conclusions
The gel technology based on lipid nanocapsules loaded with a lipophilic pro-drug of gemcitabine
injected subcutaneously were able to target the adjacent lymph nodes such as the mediastinal
lymph nodes and to combat locally the metastases by displaying an antitumor efficacy as efficient
as a treatment delivered intravenously which combat the primary tumor spreading the metastases
(i.e., Gem-C12 loaded in LNC or micelles, both delivered intravenously). Moreover, LNC loaded
with Gem-C12 and delivered iv or sc did not induce myelosuppression unlike the conventional
systemic gemcitabine hydrochloride.
20
References
1.
Siegel R, Ma J, Zou Z and Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014; 64:
9-29.
2.
IARC. 2008 [updated 11/08/2014]; Available from: http://globocan.iarc.fr.
3.
Detterbeck FC, Boffa DJ and Tanoue LT. The new lung cancer staging system. Chest
2009; 136: 260-71.
4.
Wakelee HA, Bernardo P, Johnson DH and Schiller JH. Changes in the natural history of
nonsmall cell lung cancer (NSCLC)--comparison of outcomes and characteristics in patients with
advanced NSCLC entered in Eastern Cooperative Oncology Group trials before and after 1990.
Cancer 2006; 106: 2208-17.
5.
Molina JR, Yang P, Cassivi SD, Schild SE and Adjei AA. Non-small cell lung cancer:
epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 2008; 83: 584-94.
6.
Howlader N, Noone AM, Krapcho M, Neyman N, Aminou R, Waldron W et al. SEER
Cancer Statistic Review, 1975-2008, National Cancer Institute. 2010; Available from:
http://seer.cancer.gov/archive/csr/1975_2008/.
7.
Fung SF, Warren GW and Singh AK. Hope for progress after 40 years of futility? Novel
approaches in the treatment of advanced stage III and IV non-small-cell-lung cancer: Stereotactic
body radiation therapy, mediastinal lymphadenectomy, and novel systemic therapy. J Carcinog
2012; 11: 20.
8.
Schiller JH, Harrington D, Belani CP, Langer C, Sandler A, Krook J et al. Comparison of
four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 2002; 346:
92-8.
21
9.
Toschi L and Cappuzzo F. Gemcitabine for the treatment of advanced nonsmall cell lung
cancer. Onco Targets Ther 2009; 2: 209-17.
10.
Moysan E, Bastiat G and Benoit JP. Gemcitabine versus Modified Gemcitabine: a review
of several promising chemical modifications. Mol Pharm 2013; 10: 430-44.
11.
Schroeder A, Heller DA, Winslow MM, Dahlman JE, Pratt GW, Langer R et al. Treating
metastatic cancer with nanotechnology. Nat Rev Cancer 2012; 12: 39-50.
12.
Moysan E, Gonzalez-Fernandez Y, Lautram N, Bejaud J, Bastiat G and Benoit JP. An
innovative hydrogel of gemcitabine-loaded lipid nanocapsules: when the drug is a key player of
the nanomedicine structure. Soft Matter 2014; 10: 1767-77.
13.
Ishikura H, Kondo K, Miyoshi T, Kinoshita H, Hirose T and Monden Y. Artificial
lymphogenous metastatic model using orthotopic implantation of human lung cancer. Ann
Thorac Surg 2000; 69: 1691-5.
14.
Heurtault B, Saulnier P, Pech B, Proust JE and Benoit JP. A novel phase inversion-based
process for the preparation of lipid nanocarriers. Pharm Res 2002; 19: 875-80.
15.
Calvo P, Gouritin B, Chacun H, Desmaele D, D'Angelo J, Noel JP et al. Long-circulating
PEGylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery. Pharm Res
2001; 18: 1157-66.
16.
Allard E, Passirani C, Garcion E, Pigeon P, Vessieres A, Jaouen G et al. Lipid
nanocapsules loaded with an organometallic tamoxifen derivative as a novel drug-carrier system
for experimental malignant gliomas. J Control Release 2008; 130: 146-53.
17.
Peltier S, Oger JM, Lagarce F, Couet W and Benoit JP. Enhanced oral paclitaxel
bioavailability after administration of paclitaxel-loaded lipid nanocapsules. Pharm Res 2006; 23:
1243-50.
22
18.
Hirsjarvi S, Dufort S, Gravier J, Texier I, Yan Q, Bibette J et al. Influence of size, surface
coating and fine chemical composition on the in vitro reactivity and in vivo biodistribution of
lipid nanocapsules versus lipid nanoemulsions in cancer models. Nanomedicine 2013; 9: 375-87.
19.
Hirsjarvi S, Dufort S, Bastiat G, Saulnier P, Passirani C, Coll JL et al. Surface
modification of lipid nanocapsules with polysaccharides: from physicochemical characteristics to
in vivo aspects. Acta Biomater 2013; 9: 6686-93.
20.
David S, Carmoy N, Resnier P, Denis C, Misery L, Pitard B et al. In vivo imaging of
DNA lipid nanocapsules after systemic administration in a melanoma mouse model. Int J Pharm
2012; 423: 108-15.
21.
Texier I, Goutayer M, Da Silva A, Guyon L, Djaker N, Josserand V et al. Cyanine-loaded
lipid nanoparticles for improved in vivo fluorescence imaging. J Biomed Opt 2009; 14: 054005.
22.
Bastiat G, Pritz CO, Roider C, Fouchet F, Lignieres E, Jesacher A et al. A new tool to
ensure the fluorescent dye labeling stability of nanocarriers: a real challenge for fluorescence
imaging. J Control Release 2013; 170: 334-42.
23.
Van den Broeck W, Derore A and Simoens P. Anatomy and nomenclature of murine
lymph nodes: Descriptive study and nomenclatory standardization in BALB/cAnNCrl mice. J
Immunol Methods 2006; 312: 12-9.
24.
Gemcitabine. Gemcitabine FDA-approved label. 2005 [updated 10/02/2014]; Available
from: http://www.accessdata.fda.gov/drugsatfda_docs/label/2005/020509s033lbl.pdf.
25.
Custer RP, Bosma GC and Bosma MJ. Severe combined immunodeficiency (SCID) in the
mouse. Pathology, reconstitution, neoplasms. Am J Pathol 1985; 120: 464-77.
26.
Nishioka Y and Yoshino H. Lymphatic targeting with nanoparticulate system. Adv Drug
Deliv Rev 2001; 47: 55-64.
23
27.
Trubetskoy VS, Cannillo JA, Milshtein A, Wolf GL and Torchilin VP. Controlled
delivery of Gd-containing liposomes to lymph nodes: surface modification may enhance MRI
contrast properties. Magn Reson Imaging 1995; 13: 31-7.
28.
Xie Y, Bagby TR, Cohen MS and Forrest ML. Drug delivery to the lymphatic system:
importance in future cancer diagnosis and therapies. Expert Opin Drug Deliv 2009; 6: 785-92.
29.
Chen J, Yao Q, Li D, Zhang B, Li L and Wang L. Chemotherapy targeting regional
lymphatic tissues to treat rabbits bearing VX2 tumor in the mammary glands. Cancer Biol Ther
2008; 7: 721-5.
30.
Oussoren C and Storm G. Liposomes to target the lymphatics by subcutaneous
administration. Adv Drug Deliv Rev 2001; 50: 143-56.
31.
Hirsjarvi S, Sancey L, Dufort S, Belloche C, Vanpouille-Box C, Garcion E et al. Effect of
particle size on the biodistribution of lipid nanocapsules: comparison between nuclear and
fluorescence imaging and counting. Int J Pharm 2013; 453: 594-600.
32.
Wang J, Lu Z, Gao Y, Wientjes MG and Au JL. Improving delivery and efficacy of
nanomedicines in solid tumors: role of tumor priming. Nanomedicine (Lond) 2011; 6: 1605-20.
33.
Ishikura H, Kondo K, Miyoshi T, Takahashi Y, Fujino H and Monden Y. Green
fluorescent protein expression and visualization of mediastinal lymph node metastasis of human
lung cancer cell line using orthotopic implantation. Anticancer Res 2004; 24: 719-23.
34.
Fujino H, Kondo K, Ishikura H, Maki H, Kinoshita H, Miyoshi T et al. Matrix
metalloproteinase inhibitor MMI-166 inhibits lymphogenous metastasis in an orthotopically
implanted model of lung cancer. Mol Cancer Ther 2005; 4: 1409-16.
24
35.
Leonard RC, Williams S, Tulpule A, Levine AM and Oliveros S. Improving the
therapeutic index of anthracycline chemotherapy: focus on liposomal doxorubicin (Myocet).
Breast 2009; 18: 218-24.
36.
Hawkins MJ, Soon-Shiong P and Desai N. Protein nanoparticles as drug carriers in
clinical medicine. Adv Drug Deliv Rev 2008; 60: 876-85.
37.
Shultz LD, Ishikawa F and Greiner DL. Humanized mice in translational biomedical
research. Nat Rev Immunol 2007; 7: 118-30.
38.
Galluzzi L, Senovilla L, Zitvogel L and Kroemer G. The secret ally: immunostimulation
by anticancer drugs. Nat Rev Drug Discov 2012; 11: 215-33.
39.
Ghansah T, Vohra N, Kinney K, Weber A, Kodumudi K, Springett G et al. Dendritic cell
immunotherapy combined with gemcitabine chemotherapy enhances survival in a murine model
of pancreatic carcinoma. Cancer Immunol Immunother 2013; 62: 1083-91.
40.
Yanagimoto H, Shiomi H, Satoi S, Mine T, Toyokawa H, Yamamoto T et al. A phase II
study of personalized peptide vaccination combined with gemcitabine for non-resectable
pancreatic cancer patients. Oncol Rep 2010; 24: 795-801.
25
Captions of Figures
Figure 1. A : i) Extracted organs 72h after sc injection of DiD-Gem-C12 LNC (hydrogel); ii)
Fluorescence signal in the organs (10-ms exposure at 630-800 nm); iii) Scheme of extracted
organs; iv) Regions of interest defined for the extracted organs in order to semi-quantify the
amount of photons/cm2/s. B: DiD plasmatic concentration (µg/mL) vs time in healthy nude mice,
after () iv injection of DiD LNC in suspension and () sc injection of DiD-Gem-C12 LNC
hydrogel. C to F: Biodistribution of DiD LNC in healthy nude mice, after iv injection of DiD
LNC in suspension (C and E) and sc injection of DiD-Gem-C12 LNC hydrogel (5% GemC12/Labrafac w/w) (D and F). Semi-quantification of the fluorescence signals of the removed
liver, spleen and inguinal, axillary, cervical and brachial lymph nodes (LN) using fluorescence
images (10 ms integration time) at various times post LNC administrations (1, 4, 8, 48, 96 and
336h). (n=5, mean  SD).
Figure 2. A: Kaplan-Meier survival and B: weight evoluation curves of 10 SCID-CB17 mice
grafted with 2×104 Ma44-3 cells (implanted in the left lobe) and randomized on day 0. The
negative controls were iv saline treated group (), iv non-loaded LNC treated group () and sc
non-loaded LNC treated group (). The groups were treated by gemcitabine hydrochloride ()
or Gem-C12 loaded in micelles () or loaded in LNC (), delivered by iv route; or Gem-C12
loaded in LNC (), delivered by sc route. The dose was 40 mg (molar equivalent gemcitabine
hydrochloride) per kilo of body weight beginning on day 5. The treatments were administered iv
(continuous line) via the tail vein three times a week or by sc (dashed line) twice a week.
26
Figure 3. Visualization of lungs and mediastinum of three SCID-CB17 mice at day 5 after tumor
implantation, following oral administration of 400 mg/kg of 5-aminolevulinic acid and iv
administration of DiD LNC using A: Pp IX or B: DiD visualization mode; or sc administration of
DiD-Gem-C12 LNC using C: Pp IX or D: DiD visualization mode using a CRI Maestro system.
Arrows pointed the localization of mediastinal lymph nodes in the pictures.
27
Tables
Table 1. Physicochemical characteristics of non-loaded LNC for iv and sc injection, Gem-C12
LNC for iv and sc injection, DiD LNC for iv injection and DiD-Gem-C12 LNC for sc injection.
(n=3; mean ± SD).
Gem-C12
ξ-Potc
Z-Avea
PdIb
(nm)
payload
Form
(mV)
(mg/mL)
a
Non-loaded LNC (iv)
68 ± 3
0.08 ± 0.02
-8 ± 3
---
Liquid
Non-loaded LNC (sc)
67 ± 2
0.07 ± 0.01
-10 ± 5
---
Liquid
Gem-C12 LNC (sc)
53± 2
0.07 ± 0.01
-7 ± 4
11 ± 0.2
Hydrogel
Gem-C12 LNC (iv)
53 ± 1
0.04 ± 0.01
-7 ± 3
2.75 ± 0.05
Liquid
DiD-Gem-C12 LNC (sc)
56 ± 2
0.04 ± 0.02
-5 ± 2
11 ± 0.2
Hydrogel
DiD LNC (iv)
55 ± 2
0.06 ± 0.02
-7 ± 4
---
Liquid
Z-Average, b Polydispersity index, c Zeta potential.
28
Table 2. Complete blood counts in different groups (n=5; mean ± SD, *: p < 0.05): WBC:
complete granulocyte count. RBC: red blood cell count. HGB: hemoglobin rate. HCT:
hematocrit. PLT: platelet count.
WBC
RBC
HGB
HCT
PLT
(giga/L)
(tera/L)
(g/dL)
(%)
(giga/L)
Saline iv
0.2 ± 0.1
9.8 ± 0.3
15.1 ± 0.3
47 ± 1
469 ± 50
Gemcitabine iv
0.2 ± 0.1
9.2 ± 0.3
14.0 ± 0.4
45 ± 1
245 ± 63 *
Gem-C12 iv
0.3 ± 0.2
9.1 ± 0.6
14 ± 1
45 ± 2
275 ± 63
Non-loaded LNC iv
0.6 ± 0.3
9.8 ± 0.2
15.1 ± 0.3
47.7 ± 0.9
465 ± 44
Gem-C12 LNC iv
0.14 ± 0.03
8.9 ± 0.2
13.7 ± 0.3
43.1 ± 0.9
284 ± 24
Non-loaded LNC sc
0.4 ± 0.1
9.4 ± 0.7
14 ± 1
46 ± 3
453 ± 20
Gem-C12 LNC sc
0.26 ± 0.06
9.4 ± 0.5
14.5 ± 0.8
45 ± 2
281 ± 83
29
Table 3. Biological analysis in different groups (n=5; mean ± SD, *: p < 0.05): ASAT: aspartate
aminotransferase. ALAT: alanine aminotransferase. PH ALK: alkaline phosphatase.
ASAT
ALAT
PH ALK
Creatinine
(UI/L)
(UI/L)
(UI/L)
(mg/L)
Saline iv
138 ± 115
24 ± 8
146 ± 8
1.8 ± 0.4
Gemcitabine iv
98 ± 27
27 ± 12
129 ± 17
3±3
Gem-C12 iv
152 ± 95
23 ± 8
97 ± 33 *
2±1
Non-loaded LNC iv
77 ± 20
22 ± 4
139 ± 4
1.7 ± 0.5
Gem-C12 LNC iv
82 ± 21
18 ± 7
143 ± 14
3±2
Non-loaded LNC sc
88 ± 52
24 ± 16
109 ± 5
2±1
Gem-C12 LNC sc
157 ± 52
32 ± 10
121 ± 11
4±3
30
Figures
Figure 1.
31
Figure 2.
C
32
Figure 3.
33