Delivery of mRNA by lipid-based nanoparticles enabled the development of a new generation of therapeutic agents [1
], which allowed not only the development of prophylactic vaccines for SARS-CoV-2 coronavirus, but is also in development for various other types of therapeutic intervention, including the therapy of different cancer entities [2
]. For most of these applications, delivery of the nanoparticles to certain organs or cellular compartments is required. For example, it has been shown that lipoplex (LPX) nanoparticles can be engineered for targeting either lung or spleen, depending on their physicochemical characteristics such as size and charge [5
]. In the spleen, the main target has been antigen-presenting cells (APCs), which enabled development of a technology platform for application in cancer immunotherapy, with several products currently being evaluated at different stages of clinical studies [5
]. The LPX are used for treating different cancer indications, including melanoma. Several other applications of the LPX are currently being tested in clinical studies or are in preparation (undisclosed).
Other approaches of therapeutic intervention in cancer require direct targeting of the cargo to the tumor. While such tumor targeting is already a challenge for delivery of small molecules, with mRNA as therapeutically active compound the complexity is even higher, as not only accurate control of physical targeting, but also successful uptake and translation into the encoded protein by the targeted cells must be accomplished. Depending on the intended therapeutic intervention, different cell types in the tumor microenvironment need to be targeted. In such cases, an accurate control of the target cells and a better understanding of uptake mechanisms are required for designing the RNA therapeutics.
Typically, such information can be obtained only with in vivo studies, as the usual cell culture models are not sufficiently predictive for the complex 3D cellularity in the tumor. However, the extensive screening needed to elucidate coherence between formulation characteristics and targeting at the cellular level exceed the possibilities of in vivo studies. Therefore, improved in vitro models, which are predictive for the situation in vivo, would be extremely helpful to facilitate development and screening of such pharmaceutical products.
We here addressed cell-type specific targeting, non-cell-autonomous effects and penetration capacity of mRNA lipoplex nanoparticles using the translation of the reporter protein, eGFP, after application of the LPX to 2D and 3D cell models of melanoma.
Cutaneous melanoma is a highly aggressive form of skin cancer deriving from the transformation of pigment-producing melanocytes, which are normally found in the basal layer of the epidermis [8
]. With increasing progression of the disease, radial growth phase, vertical growth phase, and metastatic melanoma can be distinguished [9
]. These three phases are characterized by lateral spread, occasional passage through the basement membrane, and dissemination through the bloodstream, respectively. Surgical removal of the tumor is only feasible until the radial growth phase and the early vertical growth phase [12
]. Treatment of subsequent stages, in general, is known for low response rate and multidrug resistance [13
The applied 3D cell model, which is composed of CCD-1137Sk fibroblasts, HaCaT keratinocytes and SK-MEL 28 melanoma cells [14
], was previously shown to recapitulate basic features of early melanoma, i.e., radial growth phase and early vertical growth phase, including loss of keratinocyte differentiation, melanoma cell invasion, and cytostatic-induced increase of ABCB5 expression in external melanoma cells [14
]. In the present study, treatment with mRNA lipoplex nanoparticles revealed distinct effects on eGFP translation levels of the different cell types, depending on the spatial 3D structure and the complexity of the systems.
2. Materials and Methods
2.1. Assembly of LPX
LPX were assembled using protocols as described earlier [5
]. Briefly, mRNA encoding for eGFP, provided in a HEPES/EDTA buffer (10 mM/0.1 mM) was conditioned with a sodium chloride solution to arrive at a sodium chloride concentration of 224 mM and a RNA concentration of 0.2 mg/mL (~0.66 mM). The RNA (unmodified) was manufactured at BioNTech (Mainz, Rhineland-Palatinate, Germany) using internal protocols. The conditioned RNA was mixed one-to-one (v
) with cationic liposomes consisting of DOTMA (R-1,2-di-O-oleoyl-3-trimethylammonium propane) as a cationic lipid and DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine) as helper lipid in a 2:1 molar ratio. Liposomes were manufactured at BioNTech with a proprietary protocol derived from the ethanol injection technique. The concentration of the DOTMA in the liposomes was 0.284 mg/mL (~0.42 mM), corresponding to a molar ratio DOTMA/RNA (calculated as one negative charge per nucleotide, 33 Da) of about 0.65. The molar (charge) ratio of DOTMA to RNA inside the LPX was about one to one [16
]. Because the LPX were assembled at an excess of RNA regarding charge ratio, an equivalent fraction of free, uncomplexed RNA (~35%) was present in the formulations. The LPX were compact globular particles and were characterized by a distinct internal lamellar organization consisting of repeating lipid bilayers where the RNA was inserted into the hydrophilic slab in between the adjacent bilayers [5
]. For quality control, the sizes of the liposomes and the LPX were determined by using dynamic light scattering measurements (Nicomp ZLS Z3000, Santa Barbara, CA, USA). Concentration of the lipids in the liposome was controlled by RP-HPLC (Agilent). Size measurements from two independent manufactured batches yielded results as follows:
| ||Size (nm)||Polydispersity index|
|Lipoplex Batch 1||264||0.14|
|Lipoplex Batch 2||266||0.12|
RNA concentrations in the starting phase and the final lipoplex formulation were determined by UV-vis measurements (Nanodrop, Thermo Fisher Scientific Inc., Waltham, MA, USA), RNA integrity was controlled by capillary electrophoresis measurements (Fragment Analyzer, Agilent Technologies Deutschland GmbH, Waldbronn, Baden-Württemberg, Germany), typical integrity values were >95%. Prior to the measurements described here, the activity of the LPX was controlled by internal standard cell culture measurements at BioNTech.
2.2. Cell Culture and Lipoplex Nanoparticle Treatment
For 2D as well as spheroid cultures, human fibroblast CCD-1137Sk (ATCC®
CRL-2703™), human keratinocyte HaCaT (CLS order no. 300493) and human melanoma SK-MEL 28 (CLS order no. 300337) cell lines were used. SK-MEL 28 and HaCaT cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (4.5 g/L), L-Glutamine, and sodium pyruvate (Capricorn, Scientific GmbH, Ebsdorfergrund, Hessen, Germany), supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 1% Penicillin Streptomycin (Capricorn, Scientific GmbH, Ebsdorfergrund, Hesse, Germany). CCD-1137Sk cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) with L-Glutamine, supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), and 1% Penicillin Streptomycin (Capricorn, Scientific GmbH, Ebsdorfergrund, Hessen, Germany). All cells were maintained at 37 °C in 5% CO2
. On day 0 of each experiment, cell viability was determined (Vi-CELL XR, trypan blue method, Beckman Coulter Inc., Brea, CA, USA). Routinely, cells were tested for mycoplasm using the MycoAlert Mycoplasm Detection Kit (Lonza Group AG, Basel, Basel city, Switzerland). All 2D and 3D experiments were performed in mono- and cocultures. Transfections with LPX nanoparticles and RNA encoding eGFP were executed in serum-free medium for a period of 24 h at 37 °C in 5% CO2
. The LPX concentration range used for transfection of the 2D cultures was based on previous reports and adapted to the different cell lines used in this study [5
]; (NCT02410733). As reference concentration for 2D cultures, 1.25 ng/µL of the LPX was used. The 3D cultures required 10 ng/µL LPX for transfection.
2.3. 2D/3D Mono- and Cocultures
The preparation of 2D cultures required 96-well flat-bottom microplates (Greiner AG, Kremsmünster, Upper Austria, Austria). For 2D mono- as well as for 2D cocultures, a total number of 3 × 104 viable cells per well (viability > 96%) were seeded. In each experiment, the cell number was determined using the Vi-CELL XR (Beckman Coulter Inc., Brea, CA, USA), trypan blue method. Then, cells were cultured for 24 h before they were washed (3 × 5 min, PBS, Sigma-Aldrich, St. Louis, MO, USA) and treated with the LPX in serum-free medium. Spheroids were generated using 96-well cell repellent plates (Greiner AG, Kremsmünster, Upper Austria, Austria). For 3D monocultures, 1 × 104 viable cells per well (viability > 96%) were seeded. After seeding, the well plates were centrifuged (5 min, 34× g) to allow for rapid spheroid formation. Then, spheroids were cultured for 48 h before treatment with LPX in serum-free medium for a period of 24 h at 37 °C in 5% CO2.
Triculture spheroids composed of CCD-1137Sk, HaCaT, and SK-MEL 28 cells were prepared in 96-well plates with a cell-repellent bottom (Greiner AG, Kremsmünster, Upper Austria, Austria) in two steps: First, 1 × 104
fibroblasts per well (viability > 96%) were seeded, centrifuged (5 min, 34× g
) and cultured for 72 h. In the second step, HaCaT (1 × 104
cells/well) and SK-MEL 28 cells (2.5 × 103
cells/well, viability > 96%) were added together and centrifuged as before. After coculturing for 48 h, the spheroids were transferred to serum-free medium and treated with LPX (PBS as negative control) for 24 h. The creation of the triculture was strictly based on [14
]. After the 24 h LPX treatment, 3D monocultures and tricultures were washed with PBS (3 × 5 min) and fixed with paraformaldehyde solution (4% in PBS, RT, 30 min). For further processing, the spheroids were transferred to 1.5 mL tubes.
2.4. Live-Cell Imaging and Optical Clearing
Immediately after incubation with LPX, 2D mono- and cocultures were washed with PBS and nuclei were stained with DRAQ5 (Invitrogen, Carlsbad, CA, USA, diluted 1:1000) for 30 min at 37 °C. Analysis of GFP fluorescence in 2D cultures used live-cell confocal microscopy (see below). Analysis of eGFP translation in spheroids was performed in fixed and optically cleared whole mounts. After fixation with paraformaldehyde solution (Carl Roth, Karlsruhe, Baden-Württemberg, Germany) (4% in PBS, RT, 30 min) the spheroids were quenched with 0.5 M glycine (Carl Roth, Karlsruhe, Baden-Württemberg, Germany) in PBS for 1 h at 37 °C. Next, they were incubated in penetration buffer (0.2% Triton X-100, 0.3 M glycine, and 20% DMSO all Carl Roth, Karlsruhe, Baden-Württemberg, Germany) in PBS for 30 min at RT. Then, spheroids were washed with PBS/1% FBS and incubated in blocking buffer (0.2% Triton X-100, 1% bovine albumin serum (BSA, Carl Roth, Karlsruhe, Baden-Württemberg, Germany), 10% DMSO in PBS) for 2 h at 37 °C. For nuclear staining, DRAQ5 was incubated overnight at 37 °C with gentle shaking. Subsequently, samples were washed 5 times for 5 min in washing buffer at RT. Finally, samples were washed with ddH2
O and refractive index was adjusted to 1.456 with Dimethyl sulfoxide (Carl Roth, Karlsruhe, Baden-Württemberg, Germany), urea (Nacalai Tesque Inc., Kyoto, Kyoto Prefecture, Japan), quadrol (Tokyo Chemical Industry, Tokyo, Tokyo Prefecture, Japan), sucrose (Nacalai Tesque Inc., Kyoto, Kyoto Prefecture, Japan) and glycerol (88%) (Carl Roth, Karlsruhe, Baden-Württemberg, Germany) for 48 h at constant RT followed by mounting on 18 well μ-slides (Ibidi GmbH, Gräfelfing, Bavaria, Germany) in the same solution. Procedures were adapted from recent protocols [17
2.5. Data Acquisition and Analysis
Micrographs were taken with an inverted Leica TCS SP8 confocal microscope equipped with Leica Application Suite X software (both Leica Microsystems CMS GmbH, Mannheim, Baden-Württemberg, Germany) and 488 nm and 633 nm lasers for GFP and DRAQ5 excitation, respectively. For all images of 2D and spheroid cultures, 5×/0.15 and 10×/0.3 Fluotar objectives were used, respectively. Images of all samples within an experiment were acquired with the same setting. Quantitative image analysis was performed using ImageJ (version 1.52p, National Institutes of Health, Bethesda, MD, USA). Therefore, the areas of eGFP fluorescence signals were threshold-adjusted to the negative control. Data were normalized to the total area of DRAQ5 signals, i.e., the eGFP-positive area was determined as a ratio of nuclear fluorescence signal area. For penetration experiments, a single optical plane taken in the center of the spheroids was analyzed. For the analysis of the ring-like regions, the ImageJ (version 1.52p, National Institutes of Health, Bethesda, MD, USA) erosion function was used on single optical planes. This allowed four areas (outer ring, mid ring, inner ring and core) to be defined that represented the spheroids from the outer border to the core. Using a threshold, the eGFP signal area was determined for each part and normalized to the area of the nuclei signal. The amount of DRAQ5 staining per microscopic field showed that the number of cells in the various conditions did not vary significantly (Figure S1
All graphs were created with GraphPad Prism (version 8.0.1, GraphPad Software, San Diego, CA, USA). The data are presented as mean ± SEM and statistically analyzed using two-way ANOVA with post hoc Tukey’s test (2D data), one-way ANOVA with post hoc Tukey’s test or Student’s t-test. p-values are indicated as * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.
There is a need for predictive in vitro model systems to allow systematic and comprehensive screening of suitable delivery vehicles [19
]. So far, cell culture models lack predictivity, presumably because the standard model systems do not adequately reflect the complex situation in the tumor. This study addressed the uptake and reporter gene expression of RNA lipoplex nanoparticles in complex in vitro melanoma tumor models, namely containing tumor and stroma cells in 2D and 3D cell cultures. By using different combinations in mono- and cocultures, the lipofection behavior was found to be dependent on the cellular as well as the 3D context.
While in adherent (2D) monocultures, LPX-mediated eGFP expression was low and did not differ significantly between fibroblasts (CCD-1137Sk), keratinocytes (HaCaT), or melanoma (SK-MEL 28) cells, transfection efficiency was significantly increased in 2D tri-cultures. According to present concepts of Warburg [20
] and Reverse Warburg effect [21
], which can be observed in 70–80% of cancers [23
], interactions between fibroblasts and cancer cells can have stimulatory effects on proliferation and metabolic interactions in different types of cancers [24
], such as melanoma [26
]. Therefore, one may hypothesize that the coculturing of fibroblasts, keratinocytes, and melanoma cells in this study affected their mutual metabolic behavior, including the intensity of cellular uptake.
Using 3D monoculture spheroids, results differed substantially from those obtained with the 2D models. While transfection efficiencies in adherent monocultures were not significantly different between the three tested cell lines, spheroids composed exclusively of SK-MEL 28 cells showed the highest transfection rate of all cell lines. Evidence for this came from both microscopic (Figure 3
) as well as cytometric analysis (Figure S3
). It cannot be excluded that this was, at least partially, due to an enhanced up-/down-regulation of uptake into SK-MEL 28 or CCD-1137Sk cells, respectively. However, penetration of the reagent into the spheroids was also likely affected; whereas SK-MEL 28 spheroids showed eGFP-positive cells throughout the spheroid width, eGFP signals were limited to the outer layers in both HaCaT and CCD-1137Sk spheroids. A likely explanation for this restriction of GFP fluorescence is that CCD-1137Sk and HaCaT cells formed rather compact and tight layers, while SK-MEL 28 cells generated more loose spheroids, which could be related to their decreased E-cadherin expression [27
]. Morphologically, this was corroborated by small and densely clustered cells in spheroids composed of CCD-1137Sk and HaCaT cells and larger cells in the case of SK-MEL 28 spheroids (Figure 3
). In this context, the differential cell sizes might hint to the discussed variations in metabolic and proliferative activity. Additionally, previous work showed that both CCD-1137Sk and HaCaT cells have a low proliferation capacity in spheroids and that proliferating cells were almost exclusively found in the outer regions of spheroids [14
]. This was confirmed by additional immunofluorescence-staining data using the proliferation marker, Ki67 (Figure S4
). In summary, location in a spheroid, cell density, and metabolic/proliferative activity might be important additional determinants of lipofection efficiency.
Finally, we observed that triculture spheroids composed of a CCD-1137Sk core, a HaCaT ring, and outer groups of SK-MEL 28 cells (originally described in [14
], confirmed in Figure S2
), showed an overall reduction in eGFP-positive cells compared to the 2D tricultures and also compared to any of the monoculture spheroids. Partially, this might be explained by the special arrangement of cells in the spheroid. Indeed, in the triculture spheroids the relative amount of the most well-transfected HaCaT and SK-MEL 28 cells was reduced in comparison to the monoculture spheroids. Furthermore, the CCD-1137Sk fibroblasts, which were already weakly transfected in the monoculture spheroids and present only in the outer cell layers, were enclosed in the triculture spheroids by several layers of HaCaT and SK-MEL 28 cells. From previous studies, it is also known that 3D cultures change cellular communication and thus also influence processes such as proliferation, protein biosynthesis, and mRNA expression [28
]. Furthermore, while in the adherent cultures soluble factors released by any of the cells were likely available for all others due to the even exposure to media, the situation in the triculture spheroids might have been different. Indeed, in these cultures, fibroblasts are known to be largely separated from keratinocytes and melanoma cells, since they form a central core [14
]. Thus, while cell–cell interactions would be expected to occur at the interface between the fibroblast core and the innermost layer of the keratinocyte ring, the interaction between fibroblasts and melanoma cells in this model are likely to be only weak via paracrine factors. Whether this contributed to the uptake behavior of the triculture spheroids, has remained elusive. In summary, the results indicate that substantial differences may be observed when using cell culture models of different complexity. Correlation with in vivo results will be necessary to allow for an assessment of the predictivity of the different model settings. The present data represent a step towards the development of such complex and indicative in vitro models. Such tools can be helpful for development of novel mRNA therapeutics, where direct delivery to the tumor is required.
In conclusion, this study allowed differences in the transfection efficiency of lipoplex nanoparticles on nonimmune cells in 2D and 3D melanoma models to be revealed. Transfection behavior of RNA nanoparticle formulations depended on several factors, such as the type of the cells, the cocultivation (interaction of different cells), as well as the 3D arrangement of the cells. In particular, while in 2D, transfection efficiency was similarly high for the different cell types—fibroblasts, keratinocytes, and melanoma cells—melanoma cells were better transfected in spheroids than fibroblasts. Furthermore, transfection was limited to outer cell layers in spheroids composed of either fibroblasts or keratinocytes, but it was rather uniform in melanoma spheroids. Finally, tricultures transfected well in 2D but poorly in spheroids, which was arguably due to the special arrangement of the different cell types in 3D. Therefore, in comparison to 2D models, the spheroid model as a platform to study 3D lipofection may provide a more realistic insight into the expected behavior in vivo.