Collagen Scaffold and Lipoaspirate Fluid - derived Stem Cells for the Treatment of Cartilage Defects in a Rabbit Model

The purpose of the present study was to assess and compare the chondroregenerative properties of PLA (processed lipoaspirate) and LAF (lipoaspirate fluid) cells, in a preclinical rabbit model of knee cartilage defect. The defects were repaired by a collagen I/III scaffold and added LAF-cells, PLA-cells or no cells, upon the study group. The results showed that collagen scaffolds seeded with LAF-derived stem cells appear to have slightly better activity and outcomes when compared to PLA-cells, in terms of cartilage regeneration.

Articular cartilage restoration by hyaline-like tissue formation still represents a challenge for surgeons and researchers [1][2][3]. Stromal stem cells (i.e. somatic multipotent precursors residing in perivascular locations of the connective stroma in adult tissues) are believed to represent a valid option for cartilage repair, due to their potential ability to express specific molecular markers, continuously release of growth factors and to differentiate into the chondrogenic lineage [1]. In particular, most of the clinical and experimental studies performed to date use bone-marrow stromal cells (BMSCs), as the physiologically reasonable candidates for chondrocyte regeneration [4][5][6]. Nonetheless, the scarcity of tissue source availability in adults, along with the relatively invasive harvesting and the reduced cellular yield makes the bone marrow an inefficient stem cell source for articular cartilage restoration. During recent years, stromal stem cells residing in the vascular-stromal fraction of the adipose tissue are being proposed as a valid alternative, due to their wider availability and comparable plastic properties. Adipose tissue (AT) is indeed an excellent source for high yield of mesenchymal stem cells (namely, adipose-derived stem cells, ASCs), with an average yield of approximately 5,000 fibroblast colony-forming units (CFU-F) per gram of AT, compared with estimates of approximately 100-1,000 CFU-F per milliliter of bone marrow [6].
Moreover, AT requires minimally-invasive harvesting procedure. However, most of the procedures based on these cells are time consuming, technically demanding, require multiple interventions and ex-vivo manipulation, thus implying high costs and risks of sample and surgical site contamination. The ideal procedure should be a one stage intervention with minimal tissue manipulation. Lately it was advocated the possibility of obtaining stem cells directly from the liquid portion of the lipoaspirate (lipoaspirate fluid, LAF), by rather simple mechanical procedures, and these cells may be theoretically useful and valuable for cell-based therapies as well as those obtained from laboratory processed lipoaspirate (PLA) [5,6]. Very few reports on the in vivo regenerative potential of LAF cells are available in current literature and no evidence exists on the differences between LAF-and PLAderived cells in promoting articular cartilage repair [6][7][8].
The purpose of the present study was to assess and compare the chondroregenerative properties of PLA-and LAF-cells seeded collagen scaffolds, in a preclinical rabbit model of knee cartilage defect. The hypothesis of the study was that LAF would promote better hyaline cartilage restoration with beneficial trophic effects due to the presence of plastic stem cells along with soluble molecules.

Experimental part Materials and methods
The present study was designed as a pilot study. All experiments were conducted in accordance with the guidelines and approved by the local Ethics Committee (Iuliu Hatieganu University of Medicine and Pharmacy from Cluj-Napoca, Romania; authorization number 340/ 02.06.2015).

Study population
Nine male Crl:KBL adult New Zealand white rabbits with a mean age of 12 months and weight of 3.75 kilograms (kg) (range 3.3 to 4.1 kg) were used in the present study. Prior to the onset of the study, all animals were quarantined and left to acclimatize for 14 days. Before and after surgery, animals were housed in standard stacked cages as one individual per cage and had access to filter tap water in bottles and pelleted food.

Cell harvesting and isolation
Adipose tissue was obtained from Hoffa pad during total knee replacement in a 55 years old male healthy human donor (who signed the informed consent stating the utilization of biological samples for research purposes), split into two aliquots and alternatively processed using either enzymatic treatment and centrifugation, or using the MyStem EVO® kit (Bi-Medica Srl, Treviolo, Italy) to separate fatty and LAF portions ( fig. 1) [6]. Cells were counted and then they were plated for isolating the adherent CD105+ cells.

Cell seeding of collagen scaffolds
To perform in-vivo implantation, the cells were detached from the plastic plates by trypsinisation as follows: culture medium was discarded and the adherent cell layer was washed with sterile PBS. Two millilitres of trypsin 0.25%/ EDTA solution were added in the culture flasks and after 2-3 min, when the cells detached, 5 mL of complete medium were added. The cell suspension was transferred into a 15 mL centrifuge Falcon tube and centrifuged at 1200 rpm for 5 min.
The cells were re-suspended in 1 mL of complete medium and counted with a Thoma haemocytometer. Sterile collagen I/III membranes (Chondro-Gide®; Geistlich Pharma AG, Wolhusen, Switzerland) of 6 mm diameter were placed in Petri dishes and 1x10 5 LAF and PLA cells were added alternatively to each plate. The cell-seeded collagen membranes were visualized in phase contrast microscopy after 24 h ( fig. 2). All the surgical procedures were performed under general anesthesia and by use of sterile conditions. The rabbits were anesthetized using diazepam with a dosage of 1 mg/kg, followed by ketamine 35 mg/kg and xylazine 5 mg/kg. A local subcutaneous infiltration with bupivacaine was also done.
A medial parapatellar incision in the right knee was done to expose the joint. With the aim of a biopsy punch needle and a drill bit, a 6-mm wide and 3-mm deep osteochondral defect was created in the femoral trochlea ( fig. 3). Animals were divided into three groups, consisting of three animals each, according to treatment: in group A (control group), the defect was filled with unseeded collagen scaffold (ChondroGide®); in group B the scaffold was seeded with LAF cells and in group C with PLA cells ( fig. 4).
In all the cases, the scaffold was fitted into the defect and glued in place with a fibrin sealant (Tisseel Lyo®; Baxter, Deerfield, IL, USA). Surgical wound closure was performed in anatomical layers, with 3-0 poliglycolic acid sutures and without drainage.
The animals received postoperative medication: tramadol hydrochloride at a dosage of 2 mg/kg each day for 7 days, meloxicam 0.5 mg/kg for 3 days and enrofloxacin 10 mg/kg for 7 days. After surgery, the rabbits were returned to their cages without any immobilization A B of the operated limb and full weight-bearing was allowed as tolerated. General health and weight-bearing status was monitored during recovery.
Outcome measurements Animals were euthanized three months after surgery, by anesthetizing them using the protocol described above and followed by an intravenous injection of potassium chloride. The distal femur was dissected and removed. With the use of a micro saw, two samples were taken from each rabbit. One sample was sunk immediately in liquid nitrogen for further molecular analysis, and the other one was fixed in 4% paraformaldehyde for morphological studies.
The main endpoints of the study were the quantitative analysis of cartilage proliferation gene expression and of macroscopic aspect according to the Wayne Cartilage Repair Score [8] (table 1). Histological qualitative evaluation and surveillance of complications were also performed, as this technique is usually used for tissue evaluation [8][9][10][11][12][13][14].
After 24-h incubation with 4% paraformaldehyde buffered in PBS at 4°C and pH 7.5, the samples were washed in water for one hour and then decalcified for 15 days dehydrated and embedded in paraffin. Tissue samples were then cut into 15-ìm thick sections using a microtome and stained with Alcian Blue for cartilage tissue analysis [15][16][17][18].
In order to evaluate the formation of cartilage components, quantitative real-time polymerase chain reaction (qPCR) was used to analyse the expression of genes involved in cartilage differentiation. To this aim, total RNA was isolated from the nitrogen frozen samples of the  [8] three groups, plated into 6-well plates, using the RNeasy MiniElute Cleanup Kit (Qiagen, Valencia, CA, USA), according to the manufacturer's instructions, as previously described [19][20][21]. An additional on-column DNAse incubation step was performed to allow the selective removal of genomic DNA during the isolation process. Thereafter, two-step reverse transcription and qPCR were carried out as previously described [19,22,23]. The resulting cDNA was used as template for qPCR, to analyse the expression of genes encoding alpha1 chains of type II collagen (COL2A1), Aggrecan (ACAN) and transcription factor SOX-9, all involved in cartilage differentiation. The 2-ÄÄCt method [23] was applied to calculate fold differences in gene expression using the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for data normalization. PCR products were subjected to melting curve analysis to rule out synthesis of unspecific products. The oligonucleotide primers were designed using Primer 3 software (http://bioinfo.ut.ee/primer3-0.4.0/).

Data analysis
Continuous outcomes were analyzed in order to assess significant differences between groups. For gene expression analysis a simple t-test was performed. For the macroscopical scoring a two-way analysis of variance (ANOVA) with Bonferroni post hoc test to compare replicate means by rows (the parameters of Wayne score: coverage, color, margins and surface) and a one-way ANOVA with Tukey's multiple comparison test to compare all columns (the means for each group), with the significance limit set at 0.05 (GraphPad Prism 7.02 Software).

Results and discussions
We encountered no important postoperative complications among the studied individuals, except for two cases with a local swelling which persisted for three weeks. The rabbits regained full function of their limb at 4 weeks, when they were able to walk without limping.

Gene expression analysis
Quantitative real time PCR -qPCR showed that the expression of the genes involved in cartilage differentiation was significantly stronger in LAF Group.
More explicit, in group B the ACAN gene expression was the highest compared to group A and C (p<0.0005). Concerning the expression of COL2A1 and SOX-9 genes in group B, it was highly statistically significant greater in comparison with group A (p<0.0005), and simply statistically significant greater when compared to group C (p<0.05) ( fig. 5).

Macroscopic evaluation
At the moment of sacrifice, we did not find any signs of local inflammation, infection or other pathological joint condition. The gross appearance seemed better for Group B, with a wider coverage of the defect by a more cartilaginous-like tissue ( fig. 6).
The overall macroscopic evaluation of cartilage repair is presented in table 2 and figure 7. The highest value of the Wayne Score was obtained in the LAF group, a mean of 11.3 out of 16 points, with the ANOVA test showing a highly significant difference among groups (p=0.0041).Tukey's multiple comparison test showed statistically significant differences between group A and group B and respectively group C, with no significant differences between group B and C (table 3).
The macroscopic evaluation showed highly significant larger cartilage surface coverage (p<0.001) and significantly smoother surface (p<0.05) in group B than in group A, and highly significant better cartilage coverage (p<0.001) and color (p<0.05) in group C comparatively to group A ( fig. 8).
We performed a qualitative analysis of the repaired tissue in the two groups with adipose derived stem cells, according to specific histologic parameters ( fig. 10). According to the evaluation of group B (LAF), we found the following: -Comparable staining, thickness and alignment of the neocartilage in both groups, with almost complete bonding to the native cartilage and tidemark continuity -More hyaline cartilage, with intact and more regular surface than in PLA group -Lesser cellularity and disorganization, with fewer chondrocyte clusters comparative to PLA group -Better reconstruction of the subchondral bone, with less fibrous tissue and almost complete bonding with adjacent bone.
These images are in agreement with gene expression analysis and macroscopic evaluation, showing the  Highly significant larger cartilage surface coverage (p<0.001) and significantly smoother surface (p<0.05) in group B than in group A, and highly significant better cartilage coverage (p<0.001) and color (p<0.05) in group C comparatively to group A.

Histologic evaluation
As we can see in figure 9.A the cartilage tissue is colored bright blue in Alcian Blue staining. The difference between cartilage tissue and bone tissue is remarkable. The cartilage tissue is rich in chondrocyte cells and matrix. Figure 9.B and 9.C show the rounded form of chondrocytes, assembled in groups of two or more, in a granular or almost homogeneous matrix.  This finding is supported by the results obtained: the LAF group presented a significantly stronger expression of cartilage proliferation genes (qPCR), the best Wayne macroscopical score (11.3 points) and ver y good histological evaluation of the regenerated cartilage and subchondral bone.
Our study was just a pilot research and we are aware of some limitations. Although rabbit is widely used as small animal model [23][24][25], the translation to clinical human model may not be as accurate. Also, the small number of animals included didn't allow us to draw statistically powerful conclusions. We could not perform a micro-CT scan on all animals, although this method offers a good visualization of the regenerated tissue.
Adipose derived stem cells (ASCs) can be isolated in similar quantity and quality from both fatty and liquid portions of the lipoaspirate. It was not the purpose of this article to describe the isolation and culture of these cells, as it has been previously described [6,9], but to provide a practical evidence of their potential in-vivo use for osteochondral regeneration.
Osteochondral regeneration by adipose derived stem cells implantation, with different types of scaffolds, is reported in literature based on various in-vivo experiments but in almost all studies, ASCs are obtained from processing the stromal-vascular fraction (SVF) of the lipoaspirate (PL A). Reports about using LAF-cells for in-vivo experiments are scarce and particularly absent those regarding cartilage repairs. In this context, our study represents a novelty, and the results encourage us to continue the research in this field. The role of cells in cartilage regeneration is incontestable. As our results show, the groups with added ASCs present statistically significant better cartilage formation than the control group, where the collagen I/III membrane was implanted without adding cells, although with this method there are very good results reported in the literature [28][29][30]. The utilization of a validated chondrogenic scaffold (Chondro-Gide®; Geistlich Pharma AG, Wolhusen, Switzerland) [31][32][33] eliminated the risk of bias due to surgical procedure and implants.
Remarkable is the highly significant superior chondrogenic gene expression in the LAF group, which could mean that their activation, differentiation and proliferation capacities are much more powerful than those of PLA-cells [34,35,36]. This is probably due to the facts that LAF contains a number of trophic molecules and tissue fractions that cooperate with stem cells in exploiting regenerative properties and, the isolation process of PLAcells by collagenase digestion could decrease their performances. Even if the overall results of LAF-cells group seem only slightly better when compared to PLA group, the most important advantage is represented by their relative ease of harvesting, with minimal manipulation.

Conclusions
ASCs represent valuable options for osteochondral repair. LAF-derived stem cells appear to have slightly better activity and outcomes when compared to PLA-cells, in terms of cartilage regeneration. LAF-cells can be separated rapidly with minimal tissue manipulation, making them more cost-effective and suitable for one-stage surgical procedures.


extfieldsofstudy:
Computer Science
Medicine

text:
Cost-effective generation of precise label-free quantitative proteomes in high-throughput by microLC and data-independent acquisition

Quantitative proteomics is key for basic research, but needs improvements to satisfy an increasing demand for large sample series in diagnostics, academia and industry. A switch from nanoflowrate to microflowrate chromatography can improve throughput and reduce costs. However, concerns about undersampling and coverage have so far hampered its broad application. We used a QTOF mass spectrometer of the penultimate generation (TripleTOF5600), converted a nanoLC system into a microflow platform, and adapted a SWATH regime for large sample series by implementing retention time- and batch correction strategies. From 3 µg to 5 µg of unfractionated tryptic digests that are obtained from proteomics-typical amounts of starting material, microLC-SWATH-MS quantifies up to 4000 human or 1750 yeast proteins in an hour or less. In the acquisition of 750 yeast proteomes, retention times varied between 2% and 5%, and quantified the typical peptide with 5–8% signal variation in replicates, and below 20% in samples acquired over a five-months period. Providing precise quantities without being dependent on the latest hardware, our study demonstrates that the combination of microflow chromatography and data-independent acquisition strategies has the potential to overcome current bottlenecks in academia and industry, enabling the cost-effective generation of precise quantitative proteomes in large scale.

Suppl. Fig. 3. Yeast protein quantification from microLC-SWATH using 29 x 16 m/z isolation windows.
A yeast tryptic digest was analysed using microLC (25 cm x 0.3 mm Triart-C18, 3 µl/min, 60 min gradient) and coupled to a TripleTOF5600 MS operating in SWATH mode by repeated injection of 10 µg yeast proteome digest (9x). Isolation windows were chosen as 29x16 m/z, with 40 ms accumulation time and a mass range of 400-850 m/z. Data was processed with Spectronaut v8.0 using SWATH libraries generated a) by sample fractionation ( frac ) or b) sample exhaustion ( exh ), c) using a publicly accessible spectral library ( Biognosys library , Spectronaut repository), or with a library generated by DIA-Umpire. Library a) allowed quantification of 1422 proteins, while 1157 proteins could be quantified using library b). The public library (c) did quantify 1118, and DIA-Umpire, that uses correlation patterns to create a library directly out of the SWATH data, 890 proteins. Spectral libraries (except the one obtained from Spectronaut repository) were generated according to Schubert et al. 1 Suppl. Fig. 4. Yeast peptide quantification from microLC-SWATH using 34 x 25 m/z isolation windows. A yeast tryptic digest was analysed using microLC (25 cm x 0.3 mm Triart-C18, 3 µl/min, 60 min gradient) and coupled to a TripleTOF5600 MS operating in SWATH mode by repeated injection of 10 µg yeast proteome digest (9x). Isolation windows were chosen as 34x25 m/z, with 100 ms accumulation time and a mass range of 400-1250 m/z. Data was processed with Spectronaut v8.0 using SWATH libraries generated a) by sample fractionation ( frac ) or b) sample exhaustion ( exh ), c) using a publicly accessible spectral library ( Biognosys library , Spectronaut repository), or with a library generated by DIA-Umpire. Library a) allowed quantification of 8824 peptides, while 6866 peptides could be quantified using library b). The public library (c) did quantify 8283, and DIA-Umpire, that uses correlation patterns to create a library directly out of the SWATH data, 6132 peptides. Spectral libraries (except the one obtained from Spectronaut repository) were generated according to Schubert et al. 1

Suppl. Fig. 5. Yeast peptide quantification from microLC-SWATH using 29 x 16 m/z isolation windows.
A yeast tryptic digest was analysed using microLC (25 cm x 0.3 mm Triart-C18, 3 µl/min, 60 min gradient) and coupled to a TripleTOF5600 MS operating in SWATH mode by repeated injection of 10 µg yeast proteome digest (9x). Isolation windows were chosen as 29x16 m/z, with 40 ms accumulation time and a mass range of 400-850 m/z. Data was processed with Spectronaut v8.0 using SWATH libraries generated a) by sample fractionation ( frac ) or b) sample exhaustion ( exh ), c) using a publicly accessible spectral library ( Biognosys library , Spectronaut repository), or with a library generated by DIA-Umpire. Library a) allowed quantification of 6598 peptides, while 5673 peptides could be quantified using library b). The public library (c) did quantify 6518, and DIA-Umpire, that uses correlation patterns to create a library directly out of the SWATH data, 4841 peptides. Spectral libraries (except the one obtained from Spectronaut repository) were generated according to Schubert et al. 1

Suppl. Fig. 6. Technical variability of yeast peptide quantification is low in microLC-SWATH-MS irrespective of data extraction
Fold change variability of 765 peptide precursors present in all data sets was compared throughout nine replicates. Median coefficients of variation are between 7.3 % and 8.4 % for libraries generated using respectively fractionation and exhaustion approach, 8.8 % for an unrelated yeast library, and 7 % for a library generated by DIA-Umpire. Spectral libraries (except the one obtained from Spectronaut repository) were generated according to Schubert et al. 1 Suppl. Fig. 7. Human peptide quantification from microLC-SWATH data.
A tryptic digest of a whole-cell protein extract from human K562 cells was analysed using microLC (25 cm x 0.3 mm Triart-C18, 3 µl/min, 60 min gradient) and coupled to a TripleTOF5600 MS operating in SWATH mode by repeated injection of 3 µg digest (6x). Data was processed with Spectronaut v8.0 using a SWATH library obtained from the SWATHAtlas repository 2 ( 10k library ), or using SWATH libraries generated by repeated analysis of HEK293 or HeLa cell extracts (Spectronaut repository). Data analysis using a rich library allows quantification of 20508 peptides, while 9256 peptides can be quantified using a HEK293 and 12272 using a HeLa library, respectively. Suppl. Fig. 8

. Technical variability of human peptide quantification is low in microLC-SWATH-MS irrespective of data extraction
Fold change variability of 6722 peptide precursors present in all data sets compared throughout the nine replicates. Median coefficients of variation of signal intensities are between 5.8 and 6.5 %, for all libraries. Suppl. Fig. 9. Direct correlation of peptide intensities between two human samples reveals no negative bias towards low intensity peptides Correlation of peptide intensities of two human samples is linear (R 2 = 0.98), and correlation is equally good for high and low intensity peptides over five orders of magnitude. Suppl. Fig. 10

. Coefficients of variation show little bias towards low intensity peptides
Coefficients of variation of peptide intensities of six human samples is low (mean 3-10 %) for peptides with intensities >1000 cps, and only marginally elevated for lower peptides (mean 8-12 %). Inset: Coefficients of variation of peptide intensities of six human samples. Suppl. Fig. 11. Library size is associated to quantification precision.
Protein identifications in a spectral library generated by sample fractionation (green; 3896 proteins) or using DIA-UMPIRE approach (violet; 853 proteins) were plotted against absolute protein abundances (log scale) reported by two independent datasets (Kulak et al. DIA-Umpire library indeed captures highly abundant proteins. Suppl. Fig. 12

. Total precursor intensities before and after batch correction
Combined intensities of all precursors varies with experimental batches, and is equalled out by batch correction. Suppl. Fig. 13. Protein fold change standard deviation is reduced by batch correction Protein fold change was calculated from three most abundant peptides of the representative ZRP1_YEAST protein quantified in two yeast strains in 9 replicates (3 batches). After batch correction, variability is considerably reduced.

Supplementary Tables
Suppl.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
Liposomes as Gene Delivery Vectors for Human Placental Cells

Nanomedicine as a therapeutic approach for pregnancy-related diseases could offer improved treatments for the mother while avoiding side effects for the fetus. In this study, we evaluated the potential of liposomes as carriers for small interfering RNAs to placental cells. Three neutral formulations carrying rhodamine-labelled siRNAs were evaluated on an in vitro model, i.e., human primary villous cytotrophoblasts. siRNA internalization rate from lipoplexes were compared to the one in the presence of the lipofectamine reagent and assessed by confocal microscopy. Results showed cellular internalization of nucleic acid with all three formulations, based on two cationic lipids, either DMAPAP or CSL-3. Moreover, incubation with DMAPAP+AA provided a rate of labelled cells as high as with lipofectamine (53 ± 15% and 44 ± 12%, respectively) while being more biocompatible. The proportion of cells which internalized siRNA were similar when using DMAPAP/DDSTU (16 ± 5%) and CSL-3 (22 ± 5%). This work highlights that liposomes could be a promising approach for gene therapy dedicated to pregnant patients.

Introduction
Surveys of the world's clinical trial registries showed pregnant patients are still under-represented during medication development, and particularly in evaluation and safety trials [1,2]. The routine exclusion of pregnant women from clinical trials and the absence of post-marketing surveillance are leading to a huge lack of information regarding drug efficiency and safety for both the fetus and mother [3]. In addition, pregnancy disorders, like preeclampsia, placental insufficiency, viral infection, trophoblastic cancer, and fetal growth restriction, remain without curative treatment and cause considerable health issues [4]. There is, thus, a critical need to encourage research projects aiming at providing additional preclinical safety data and developing new therapeutic strategies. More specifically, drug delivery approaches could carry new active molecules to the placenta, an organ involved in many pregnancy disorders.
Nanomedicine is expected to increase the distribution specificity of therapeutic molecules while reducing their off-target distribution therefore improving therapeutic efficacy and safety [5,6]. The advantages displayed by nanocarriers appear well-suited for pregnancy disorder treatments. Rationally designed nanocarriers could meet the essential requirements: providing an optimized treatment for the mother while avoiding side effects for the fetus by limiting the drug's transplacental passage [7].
Few reports have been published about the application of nanomedicine for the treatment of pregnancy disorders. We listed and discussed in a recent review the potential of nanomedicine, and particularly liposomes, to empower new strategies for the treatment of preeclampsia, one of the most severe pregnancy complications often responsible for prematurity [7]. We recently reported that PEGylated liposomes penetrate the outer cell layer of the placenta without gaining access to the fetal circulation [8]. This finding support the potential of liposomes as carriers of therapeutic molecules, including gene therapy agents, such as small interfering RNA (siRNA).
RNAi-based medicine holds a pivotal place in the future treatment of human diseases. siRNA are double-stranded RNA oligonucleotides able to induce the silencing of a target gene via RNA interference. Specificity provided by the siRNA sequence could be exploited in the development of therapeutic strategies for pregnancy disorders [9]. However, their instability in biological fluids, as well, as their hydrophilicity, hinder delivery to the target tissue and cellular uptake. Liposome-based formulations could offer the opportunity (i) to protect fragile nucleic acids from nucleases degradation; (ii) to target siRNA delivery to the outer cell layer of placenta villi (the syncytiotrophoblast); and (iii) to restrict off-target effects especially towards the fetus.
In this work we evaluated the cellular internalization of siRNA carried by three different liposome-based formulations. All three formulations are designed to reach neutrality after siRNA loading and have previously been shown to favor transfection with a limited toxicity on other cell types [10][11][12]. Formulations complexed with a fluorescently-labelled siRNA were incubated in vitro with primary human villous cytotrophoblasts. Cells were then observed using confocal microscopy in order to estimate the rate of siRNA internalization into the cells.

Formulation Characterization
In this study, we selected three formulations previously developed for gene delivery purposes aiming at avoiding the well-known toxicity associated to cationic lipoplexes. The preparation methods and compositions were used as described in [10][11][12], and they are based on the addition of specific lipids and polymers. DMAPAP is a lipopolyamine chosen from a library of cationic lipids and used in our laboratory for its reduced toxicity and high reproducibility as an in vitro transfecting agent [13]. DDSTU is a lipothiourea designed to interact with nucleic acids through hydrogen bond interactions, providing high cargo release efficacy and low toxicity [14]. CSL-3 is a pH-dependent switchable lipid able to complex siRNA and to release it following a pH change [15]. DMAPAP+AA formulation contained 50% of DMAPAP and 5% of PEGylated lipid. An anionic polysaccharide, sodium alginate (AA), was added concurrently to siRNA when preparing DMAPAP+AA, to increase the silencing efficiency and decrease cellular toxicity. DMAPAP/DDSTU formulation was composed of 25% of DMAPAP, 70% of DDSTU, and 5% of PEGylated lipid; mixing DMAPAP and DDSTU is expected to bring both internalization and release abilities. The CSL-3 formulation contained 50% of CLS3 as the cationic lipid and 2.5% of PEGylated lipid. Complete compositions and preparation methods are described in the Materials and Methods (see Sections 4.1 and 4.2).
Physicochemical characterization was performed with or without siRNA, referred to as lipoplexes and liposomes, respectively. The hydrodynamic diameter, polydispersity index (PdI), zeta potential, and encapsulation rate are shown in Table 1. The hydrodynamic diameters of the DMAPAP-based formulations were around 100 nm, while the diameters of CSL-3-based formulations were around 170 nm. After siRNA complexation, a size increase was observed for all three formulations. Zeta potentials of all formulations were moderately positive and reached neutrality after siRNA addition due to electrostatic interactions between cationic liposomes and siRNA, indicating complexation. Complexation was confirmed and quantified by RiboGreen ® assay. The results showed that the siRNA was not available for the fluorescent probe, indicative of a full complexation of siRNA in the three nanoconstructs.

siRNA Delivery to Primary Cytotrophoblasts
The placenta is the interface organ connecting maternal and fetal circulations and controlling the passage of gas, nutrients, and xenobiotics. The outer cell layer, the syncytiotrophoblast (ST), is in direct contact with the mother's blood flow containing the administered drugs. The culture of human primary villous cytotrophoblasts (VCT) is the most relevant in vitro model in the context of the formulation evaluation for pregnancy-related disease therapy. Indeed, the VCT differentiates in vitro to form a functional syncytiotrophoblast by cell fusion.
To evaluate the delivery efficiency of the three formulations, villous cytotrophoblasts were incubated with formulations loaded with rhodamine-labelled siRNA ( Figure 1). Lipofectamine, a commercial cationic lipid-based reagent, is commonly used for in vitro cells transfection, including villous cytotrophoblasts [16]. Lipofectamine was used in this study as a delivery-positive control. Due to the toxicity of lipofectamine, incubation time must be limited to a maximum of 5 h. Confocal microscopy observations showed that all tested formulations led to fluorescent siRNA uptake in the primary cytotrophoblasts. The observed rhodamine fluorescence intensity in the cells incubated with the three formulations for 24 h is visually comparable to the one obtained with lipofectamine after 5 h. Therefore, the three tested formulations are able to deliver siRNA to human primary cytotrophoblasts.
In order to compare the formulations, a semi-quantitative analysis was performed on confocal images. The percentage of VCT internalizing siRNA was calculated based on the detection of the rhodamine signal into cells marked with cytokeratin 7, a specific marker of human trophoblasts ( Figure 2).

Discussion
In this paper, we report for the first time siRNA internalization into human primary cytotrophoblasts using liposomes as nanovectors. The tested formulations were previously developed and designed for in vivo applications. Results showed that DMAPAP+AA, DMAPAP/DDSTU, and CSL-3 had similar or lower delivery efficiency compared to the commercial reagent, lipofectamine. In addition, these formulations are serum compatible allowing an extended incubation time to 24 h in comparison to lipofectamine (5 h maximum). A high tolerance of the lipoplexes formulations was previously reported in vitro with cells from non-placental origin [10][11][12]. Results showed that 44 ± 12% of the VCT were labelled following incubation with siRNA-lipofectamine. Association of siRNA with DMAPAP+AA led to 53 ± 15% of labelled cells, while DMAPAP/DDSTU led to 16 ± 5% and CSL-3 led to 22 ± 5% of labelled cells, respectively. The rate of labelled cells was significantly higher with DMAPAP+AA and with lipofectamine than the control. With DMAPAP/DDSTU and CSL-3, siRNA presented a similar cellular internalization rate.

Discussion
In this paper, we report for the first time siRNA internalization into human primary cytotrophoblasts using liposomes as nanovectors. The tested formulations were previously developed and designed for in vivo applications. Results showed that DMAPAP+AA, DMAPAP/DDSTU, and CSL-3 had similar or lower delivery efficiency compared to the commercial reagent, lipofectamine. In addition, these formulations are serum compatible allowing an extended incubation time to 24 h in comparison to lipofectamine (5 h maximum). A high tolerance of the lipoplexes formulations was previously reported in vitro with cells from non-placental origin [10][11][12]. The biocompatibility was also confirmed upon intravenous injection on various in vivo mice models, no sign of hemolysis was observed [10,12,17]. Further experiments to study the toxicity of lipoplexes on VCT should be performed.
Various in vitro evaluation models have been set up for the study of placental functions. Many placental cell lines, like BeWo (from choriocarcinoma origin) or HTR8 (immortalized extravillous cytotrophoblasts), can be used, but have a restricted ability to express all necessary functions and/or fail to form a functional and physiological syncytium. To overcome these limitations, the use of human primary villous cytotrophoblasts purified from human placentae seems to be the most suitable to evaluate the potency of different siRNA carriers. This model presents the advantage of both expressing a human phenotype and being representative of morphological and functional differentiation of trophoblastic cells. In addition, villous cytotrophoblasts fuse in vivo to form the syncytiotrophoblast layer which is in direct contact with the maternal circulation. A syncytiotrophoblast is obtained in vitro by the fusion of primary VCT [18]. Moreover, no interspecies data extrapolation is needed thanks to the human origin of this model. Primary cells are difficult to transfect by non-viral vectors. Fused cells, such as syncytiotrophoblasts with specific barrier properties, were not expected to efficiently allow non-viral vectors to be internalized and deliver their content. Actually, numerous transfection reagents have been tested on placental primary cells and showed highly variable levels of internalization efficacy and reproducibility [16].
In our first study, we showed that liposomes are good candidates as vectors for placental drug delivery thanks to their interaction with the syncytiotrophoblast without the transfer of formulation components to the fetus [8]. In this study, we demonstrate that siRNA loaded into lipoplexes are internalized by VCT. In a previous report, Bajoria et al. studied the internalization of liposomes loaded with a small hydrophilic molecules on human trophoblasts. The authors showed that liposomes were internalized by an energy-dependent pathway and suggested endocytosis as one of the placental cells uptake mechanisms [19]. The internalization pathway for the lipoplexes should be investigated, with endocytosis being highly probable, as this mechanism was previously reported for lipoplexes internalization in other cell types [20].
Moreover, another step in developing these new drug delivery strategies for pregnancy diseases will be to study the efficiency of gene silencing using a therapeutic siRNA. Indeed, nucleic acid internalization is only a part of the complex gene delivery process and does not necessarily imply knock-down of the gene of interest. A potential correlation between cellular internalization and transfection efficiency is however probable with the tested formulations as in vivo gene silencing was demonstrated for two of them in previous investigations [10,12,17].
Further experiments are needed to evidence the absence of placental in-depth penetration and transplacental passage of the siRNA using complementary models, like the human placental explants and the human perfused placenta.
To conclude, our results show that well designed lipoplexes are prone to deliver siRNA into the syncytiotrophoblast, a key target site in many pregnancy-related pathologies. The development of safe and efficient formulations require more investigations and more knowledge in both nanomedicine and pregnancy pathologies.
Briefly, for DMAPAP+AA, lipids (DMAPAP/DOPE/Dodagly-PEG 50/45/5, molar ratio) were dissolved in chloroform and the organic solvent was evaporated to form a thin film. The dried film was hydrated with 150 mM NaCl and extruded to obtain a homogenous suspension of liposomes (2.5 mM). For the DMAPAP/DDSTU formulation, liposomes were prepared by the ethanolic injection method with the specific molar ratio: DMAPAP/ DDSTU/Dodagly-PEG, 25/70/5, in order to obtain a final lipid concentration of 2.5 mM in 150 mM NaCl. For the CLS3-50 formulation, liposomes were prepared using the thin film method with a lipid composition and molar ratio of CSL3/DSPC/cholesterol/Dodagly-PEG 50/10/37.5/2.5. The dried film was hydrated with 5% dextrose, sonicated and extruded, and the final concentration was adjusted to 2.5 mM. Formulation preparation methods and/or hydration buffer differ from one-another as we intended to follow precisely the protocols described in [10][11][12].
Lipoplexes were prepared by mixing an equal volume of the siRNA solution (plus sodium alginate for the DMAPAP+AA formulation) to the cationic liposome suspension and rapidly mixed by vortexing. When using lipofectamine 2000, the same steps were followed, and the diluted transfection reagent was mixed with an equal volume of siRNA solution. According to the manufacturer's instructions, ratio was 1 µL of lipofectamine for 5 pmol of siRNA. Lipoplexes were incubated for 30 min at room temperature for the DMAPAP+AA and DMAPAP/DDSTU formulations and lipofectamine, 10 min at 50 • C for the CSL-3 formulation. The charge ratio was N/P = 4 for DMAPAP+AA, DMAPAP/DDSTU, and CSL-3, and was calculated based on the molar ratio of positive charges (three positive charges per molecule of DMAPAP, one per molecule of CSL3) and the molar ratio of negative charges (from siRNA and sodium alginate molecules, 3.08 nmol negative charges per µg and 5.05 nmol negative charges per µg, respectively).

Liposome and Lipoplex Characterization
Mean particle hydrodynamic diameter (Z-average), polydispersity index (PdI), and zeta potential were determined using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). Before each measurement, an appropriate dilution (1/100) of liposomes or lipoplexes suspensions was performed in NaCl 150 mM. The encapsulation efficiency of siRNA in the formulations was determined by RiboGreen ® assay according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA).

Placentae Collection
Placentae were collected from women with normal pregnancies, following caesarean section (between 37 and 41 weeks of gestation). Biological samples were obtained after informed patient written consent and approval from our local ethics committee (CCP-2015-Mai-13909).

Primary Villous Cytotrophoblasts Isolation and Purification
Primary villous cytotrophoblasts (VCT) isolation by differential trypsin digestion was performed as previously described [23]. Chorionic villi were washed, dissected, and rinsed in Ca 2+ , Mg 2+ -free HBSS, then incubated sequentially with trypsin. Each time, the supernatant containing VCT was collected after tissue sedimentation, filtered (100 µm), and incubated with 10% FCS (v/v) to stop trypsin activity. After Percoll gradient fractionation, cells were diluted to a concentration of 1 × 10 6 cells/mL in DMEM supplemented with 10% FCS, 2 mM glutamine, 100 UI/mL penicillin, and 100 µg/mL streptomycin. Cells were then plated at a density of 150,000 cells/cm 2 on 60 mm culture dishes. VCT were incubated overnight in 5% CO 2 at 37 • C and washed three times with PBS to eliminate non-adherent cells. Culture purity was assessed by immunolabelling with anti-CK7 antibody (a trophoblast-specific marker). The quality of the culture and formation of the ST was monitored by hCG quantification in cell supernatants.

Confocal Microscopy
After 48 h of primary culture, VCT were fixed in 4% paraformaldehyde for 20 min, then paraformaldehyde was neutralized with NH 4 Cl 50 mM for 10 min. Cells were rinsed three times with PBS and permeabilized in Triton 0.1% for 10 min. Saturation of unspecific sites was performed in PBS with 1% BSA and 0.1% Tween at room temperature. Primary antibodies against cytokeratin 7 were added for 2 h at room temperature and amplified with the Alexa Fluor 488 ® -conjugated secondary antibody for 2 h at room temperature. Nuclei were then stained with DAPI. Cells were mounted in a fluorescent Dako mounting medium (Dako, Glostrup, Denmark), examined, and photographed on a TCS SP2 confocal microscope (Leica, Wetzlar, Germany) of the Cellular and Molecular Imaging facility, INSERM UMS 025-CNRS UMS 3612, Faculté de Pharmacie de Paris, Université Paris Descartes, Paris, France.
Semi-quantitative assessment was performed counting at least 100 cells per condition per placentae. Statistical tests were performed using GraphPad Prism (GraphPad Software Inc. 5.01, La Jolla, CA, USA).