Combinatory Delivery of Etoposide and siCD47 in a Lipid Polymer Hybrid Delays Lung Tumor Growth in an Experimental Melanoma Lung Metastatic Model

This study investigated the feasibility of lipid polymer hybrid nanoparticles (LPH) as a platform for the combinatorial delivery of small interfering RNA (siRNA) and etoposide (Eto). Different Eto loaded LPH formulations (LPH Eto) are prepared. The optimized cationic LPH Eto with a particle size of 109.66 ± 5.17 nm and Eto entrapment efficiency (EE %) of 80.33 ± 2.55 is used to incorporate siRNA targeting CD47 (siCD47), a do not eat me marker on the surface of cancer cells. The siRNA‐encapsulating LPH (LPH siNEG‐Eto) has a particle size of 115.9 ± 4.11 nm and siRNA EE % of 63.54 ± 4.36 %. LPHs improved the cellular uptake of siRNA in a dose‐ and concentration‐dependent manner. Enhanced cytotoxicity (3.8‐fold higher than Eto solution) and high siRNA transfection efficiency (≈50 %) are obtained. An in vivo biodistribution study showed a preferential uptake of the nanosystem into lung, liver, and spleen. In an experimental pseudo‐metastatic B16F10 lung tumor model, a superior therapeutic outcome can be observed in mice treated with combinatory therapy. Immunological studies revealed elevated CD4+, CD8+ cells, and macrophages in the lung following combinatory treatment. The study suggests the potential of the current system for combinatory chemotherapy and immunotherapy for the treatment of lung cancer or lung metastasis.


Introduction
Under normal physiologic conditions, cellular homeostasis is governed by the balance between pro-phagocytic signals and DOI: 10.1002/adhm.202001853 anti-phagocytic signals. [1] In general, tumors express high levels of CD47 as a strategy to evade the clearance by the mononuclear phagocytic system (MPS). The interaction between CD47 and its signal regulatory protein (SIRP ) on the macrophages delivers a "don't eat me signal" which hinders their recognition by the immune system. [2] Blocking CD47 can be achieved by using either monoclonal antibodies [2] or small interfering RNA (siRNA). [3] Interestingly, exploiting siRNA has many advantages over antibodies including ease of manufacturing and simple incorporation into different nanocarriers with possible tumor accumulation by the enhanced permeation and retention (EPR) effect.
Combination therapy targeting multiple pharmacological mechanisms is found to improve therapeutic outcomes in cancer. [4] Etoposide (Eto) is a potent topoisomerase II inhibitor affecting mainly S and G2 phases of the cell cycle. [5] It has been explored in the treatment of many types of tumors such as lung cancer, leukemia, non-Hodgkin's lymphoma, neuroblastoma, and gastric cancer. [6] Although Eto is broadly considered to be immunosuppressive, [7] a previous study has reported its combination with an immunotherapeutic agent can lead to complete tumor regression in immunocompetent hosts. [8] A clinical study has demonstrated improved survival in lung cancer patients receiving atezolizumab, a monoclonal antibody against PD-L1, programmed death-ligand 1, with Eto and carboplatin. [9] In a preclinical study, Eto therapeutic efficacy was also augmented when combined with a blocker of CTLA-4, a T-cell inhibitory receptor. [10] Thus, the combined efficacy of an anti-proliferative chemotherapeutic drug and immunogenic activation resulting from the co-delivery of Eto and siRNA against CD47 (siCD47) is speculated to result in a potent anti-tumor activity. There is however a need to design a delivery system capable of simultaneous delivery of the chemotherapeutic agent Eto, a hydrophobic low molecular weight drug, and the hydrophilic polyanionic siRNA.
Lipid polymer hybrid nanoparticles (LPH) are nanocarriers composed of an internal polymeric core enclosed by an outer lipid shell composed of one or more layers. [11] By virtue of its unique structure, LPH has versatile competence to encapsulate different types of payloads such as hydrophilic and hydrophobic Scheme 1. The two LPH formulations prepared. Two types of LPH are prepared in the study. A) I-LPH (I refers to in situ) is prepared by dissolving a lipid mixture of lecithin: tristearin (1:1) (1 mg mL −1 ), DSPE-PEG 2000 (1 mg mL −1 ), Tween 80 (10 mg mL −1 ), stearyl amine (10 mg mL −1 ) with/or without siRNA (0.133 mg mL −1 ) in 4% hydroalcoholic solution (0.9 mL) and heated at 70°C. PLGA (5 mg mL −1 ) and Eto (3 mg mL −1 ) are dissolved in DMF (0.1 mL). The hydroalcoholic lipid phase is titrated by the drug-polymer organic solution dropwise with magnetic stirring at room temperature for 2 h. B) P-LPH (P refers to post) are prepared by omission of siRNA from the aqueous phase and the formed LPH containing Eto are subsequently mixed with siRNA solution (at a final concentration of 0.133 mg mL −1 ) for 2 h at room temperature. For both LPHs, the organic to aqueous phase is kept at 1:9 v/v volume ratio and the fabricated LPH is obtained by ultrafiltration using Amicon tubes (MWCO 100 K, 12 000 rpm for 30 min at 4°C) to separate the unentrapped Eto and/or siRNA. The collected pellets (LPH) on the membrane are re-dispersed in PBS (pH 7.4, RNAse free water) to a final volume of 1 mL for further analysis.
drugs or nucleic acids. [12,13] The concomitance of both polymer and lipid imparts the LPH with structural integrity, serum stability, sustained drug release characteristics controlled by the polymeric core, and the high biocompatibility offered by the lipid shell. [14] Previous literature has reported the feasibility of LPH for the simultaneous delivery of both chemotherapeutic agents and siRNA in different tumor models, including delivery of cisplatin prodrug and REV1/REV3L-specific siRNAs for prostate cancer [15] and HIF1a siRNA and gemcitabine for pancreatic cancer. [12] The aim of this study was to design an LPH system suitable for the co-delivery of Eto and siCD47 for in vivo delivery and anticancer therapy in a murine tumor model. For this purpose, various formulations and process parameters were optimized using Box-Behnken design (BBD). The optimized formulae were those with the maximum entrapment efficiency (EE %) and minimum particle size (<150 nm). The ability of the optimized LPH formulations to improve siRNA uptake and gene silencing was investigated in mouse melanoma B16F10 cells. The therapeutic efficacy of the LPH co-loaded with siCD47 and Eto was investigated in an experimental mouse melanoma lung metastasis model.

Preparation of LPH
The single-step nanoprecipitation self-assembly method was adopted to fabricate different types of LPH systems. [16] The preparation schemes are illustrated in Scheme1. Two strategies were attempted to introduce siRNA into the formulation. In one method, siRNA was dissolved in the aqueous phase prior to mixing with the organic phase. The LPH prepared by this method www.advancedsciencenews.com www.advhealthmat.de was referred to as I-LPH (I refers to in situ). In the second approach, referred to as P-LPH (P stands for post-incorporation), siRNA was complexed with the previously formed LPH.
To prepare I-LPH, the organic phase (0.1 mL) was prepared by dissolving PLGA (5 mg mL −1 ) and Eto (3 mg mL −1 ) in DMF. A 4 % v/v hydroalcoholic solution (0.9 mL) containing lecithintristearin (1 mg mL −1 ), DSPE-PEG 2000 (1 mg mL −1 ), Tween 80 (10 mg mL −1 ), stearyl amine (10 mg mL −1 ), and siRNA (0.133 mg mL −1 ) was prepared and heated at 70°C for 15 min. The organic phase was added slowly to the aqueous solution under 900 rpm magnetic stirring for 2 h at room temperature. The organic to aqueous phase was kept at a 1:9 v/v volume ratio. The PLGA, lecithin-tristearin and Eto concentrations stated above were verified to yield optimal formulation characteristics by BBD design described in the next section. To prepare P-LPH, LPH were firstly prepared using the same formula as I-LPH but without the siRNA. The formed LPH containing Eto (1 mL) was subsequently mixed with siRNA solution (0.1 mL) (to a final concentration of 0.133 mg mL −1 ) for 2 h at room temperature. The unentrapped Eto, siRNA, and traces of organic solvent were removed by ultrafiltration using Amicon Ultra filters (MWCO 100 K, 12 000 rpm for 30 min at 4°C). The recovered concentrated LPH was re-dispersed in PBS (pH 7.4, RNAse free water) to a final volume of 1 mL for further analysis. Eto and siRNA containing LPH were referred to as LPH Eto and LPH siNEG , respectively. LPH siNEG-Eto contains both siRNA and Eto. siNEG was replaced with siCD47 for therapeutic studies, that is, LPH siCD47 . For the biodistribution study, DiR-labeled LPH siNEG was prepared by dissolving DiR in the alcoholic aqueous phase at 1 % w/w of the total lipid.

Experimental Design and Construction of BBD
Design-Expert software (Design-Expert 9.0.5.2, State-Ease Inc., USA) was used to construct a matrix and to explore both the response surfaces and the statistical models to optimize LPH Eto formulation. The selected critical process parameters (CPPs) were divided into formulation variables namely PLGA (A), lecithintristearin (B), and Eto (C) concentrations; processing variable namely the stirring speed (D). The independent variables A-D were tested at three levels: low (−1), medium (0), and high (+1). The particle size (Y1) and EE % (Y2) were the critical quality attributes (CQAs). The LPH was designed to deliver quality target product profile (QTPP) of minimum particle size and maximum EE %. The defined CPPs and CQAs, as well as the desired QTPP, are listed in Table S1, Supporting Information. The design matrix constructed by the software involved 29 different formulae (Table  S2, Supporting Information).
The statistical validation of the polynomial equations generated by Design-Expert software was performed by ANOVA. Various statistical indices such as P-values, F-values, R 2 (adjusted R 2 and predicted R 2 ), and predicted residual error sum of squares (PRESS) were used to assess the statistical significance of the models; either linear two-factor interactions (2FI) or quadratic models. The 3D response surface plots were constructed by the software and the polynomial equations were authenticated. Different feasibilities were conducted over the experimental domain to find the compositions of the optimized LPH. Based on the highest desirability, the design space was generated to define the optimum CPPs to prepare LPH with the desired QTPP. [17] One optimum LPH Eto checkpoint was elected to confirm the chosen domain and equations. The experimental values of the responses were quantitatively compared with the predicted values and the prediction errors (%) were calculated.

Determination of Particle Size, Size Distribution, and Zeta Potential
The particle size (z-average), size distribution expressed as polydispersity index (PDI), and surface charge (zeta potential) of the prepared LPH Eto with or without siRNA (n = 3) were assessed by dynamic light scattering (DLS) with a Nanosizer ZS Series (Malvern Instruments, Southborough, MA). The as-prepared LPH dispersions were diluted with deionized water (1:10 v/v) and transferred to disposable plain folded capillary Zeta cells. All the measurements represent the average of 20 runs; each run was completed in triplicate at 25°C.

Determination of Encapsulation Efficiency (EE %) and Loading Efficiency (LE %)
The EE % was measured directly by quantifying the amount of Eto encapsulated into the LPH (n = 3). Briefly, Eto LPH was centrifuged at 12 000 rpm for 30 min at 4°C and the recovered pellets were dissolved in DMF (10 mL). Eto amount was quantified using a previously reported HPLC method. [18] The HPLC system was composed of an Agilent 1100 system, equipped with G 1311A quaternary pump and a UV detector (VWD-G1314 A). A reversephase C8 column (Thermo BDS, 250 × 4.6 mm, 5 µ) was used for Eto separation at 25°C at 210 nm detection wavelength. The mobile phase consisted of acetonitrile and 10 mM phosphate buffer pH 3.5 at 70:30 ratio, pH was adjusted to 4.0 ± 0.1 with formic acid at 1 mL min −1 flow rate.
The EE% was calculated using the following equation :

Transmission Electron Microscopy (TEM)
The optimized LPH Eto was visualized using transmission electron microscope (TEM, Jeol, JEM-1230, Japan). A drop of the LPH www.advancedsciencenews.com www.advhealthmat.de dispersion was deposited on a copper 300-mesh grid, coated with carbon, and allowed to stand for 10 min after which, any excess fluid was absorbed with a filter paper. The sample was negatively stained with one drop of 1 % phosphotungstic acid, applied, and allowed to dry for 5 min.

In vitro release study
Eto release from the optimized LPH Eto formulation was studied using the dialysis membrane method with slight modifications. [19] An aliquot volume of the prepared LPH (1 mL equivalent to 2.3 mg Eto) (n = 3) was placed in the pre-soaked dialysis membrane (10 K Da MWCO) and diluted with 1 mL of PBS (pH 7.4), mixed with FBS (at 50 % v/v concentration). The sealed membranes were dialyzed against 50 mL PBS (pH 7.4) containing 1% Tween 80 in a thermostatically controlled shaking water bath at 250 strokes min −1 ±0.1 at 37 ± 0.5°C. [20] To exclude the possible nonspecific binding of Eto to the dialysis membrane, the in vitro release of Eto solution in PBS (containing 10 % DMSO) was also conducted under the same conditions. At predetermined time intervals, an aliquot of 1 mL of the release media was withdrawn and immediately replenished with an equivolume of preheated release media. Eto concentration was quantified by measuring the absorbance at 285 nm using a UV spectrophotometer (Perkin-Elmer Lambda 35) using dialysate from drug-free LPH as a blank.

In Vitro Hemolytic Assay
In vitro hemolytic activity of the optimized LPH Eto was assessed using fresh BALB/c mouse red blood cells (RBCs) (n = 3). In brief, blood was withdrawn from BALB/c mice by cardiac puncture in heparinized tubes and the RBCs were collected by centrifugation at 4000 rpm for 10 min. Consequently, the obtained RBCs were incubated with different concentrations of Eto, free or encapsulated into LPH, for 2 h at 37°C. The samples were centrifuged at 4000 rpm for 5 min at 4°C. The amount of the liberated hemoglobin in the supernatant was quantified spectrophotometrically at 545 nm. The respective negative and positive control were prepared by incubating RBCs with PBS (pH 7.4) and Triton X-100 (0.5 % w/v), respectively. [21] Percentage (%) hemolysis was calculated using the following equation: % Hemolysis = absorbance sample − absorbance negative control absorbance positive control − absorbance negative control (3)

Cytotoxicity Study
In vitro cytotoxicity of Eto formulations was assessed using MTT assay. In brief, B16F10 cells were seeded in 96-well plates (n = 5) at a density of 7 k cells per well in RPMI media for 24 h. Subsequently, cells were incubated with serial concentrations of Eto in the range of 0.01-100 µM or the blank formulation at equivalent concentrations for 48 h. The incubation media was then replaced with MTT solution (120 µL) and cells were kept at 37°C and 5 % CO 2 for 4 h. The obtained formazan crystals were dissolved in 200 µL of DMSO then the plate was read at 570 nm using FLU-Ostar Omega plate reader (BMG Labtech). Cytotoxicity was expressed as the percentage cell survival which was calculated using the following equation: % Cell survival = absorbance of treated cells at 570 nm absorbance of untreated cells at 570 nm × 100 (4)

Cellular Uptake Study
Cellular uptake of siRNA was assessed by flow cytometry by incubating B16F10 cells with 30 nM or 90 nM of the fluorescently labeled siRNA, Atto 655-siRNA, in free or LPH form with/without Eto (0.1 µM), for 4 and 24 h (n = 3 for each condition). Cells were washed twice with PBS, harvested and centrifuged at 1750 rpm for 3 min at 4°C. Uptake was quantified by measuring the fluorescence of 10000 gated cells using FL4 detector (BD FACS Calibur flow cytometer, BD Biosciences). Analysis was performed using Flowjo software (Treestar).

In Vitro Gene Silencing
The gene silencing ability of different LPH siCD47 formulations (or their LPH siNEG counterparts) on B16F10 cells was investigated by incubation with 10, 30, and 90 nM siRNA for 48 h with/without Eto (0.1 µM) (n = 3). Cells were washed twice with PBS, trypsinized, and then stained with anti-mouse CD47-APC monoclonal antibody. CD47 expression was quantified by measuring the fluorescence of 10 000 gated cells using FL4 detector (BD FACS Calibur flow cytometer, BD Biosciences). Analysis was performed using Flowjo software (Treestar). Transfection efficiency was calculated as percentage reduction in mean fluorescence intensity (MFI) relative to control.

In Vitro Serum Stability
The effect of incubation with serum (50 % v/v FBS) on different LPH siNEG-Eto particle size, PDI, and zeta potential after 4, 24, and 48 h incubation at 37°C were studied (n = 3).

RNA Protection Test
The ability of LPH to protect the entrapped siRNA from FBS or RNAse was investigated using agarose gel retardation assay.
Adv. Healthcare Mater. 2021, 10,2001853 LPH formulations and naked siRNA, used as a positive control, were incubated with FBS (50 % v/v) or RNAse (100 µg mL −1 ) for 4, 24, or 48 h. At the end of the incubation period, RNAse was deactivated with EDTA at 50 mM final concentration and the complex was challenged with heparin (100 IU mL −1 , 10 % v/v) to dissociate siRNA from the complex. Untreated siRNA was used as a negative control. Samples were mixed with 6X loading dye and resolved on 2 % w/v agarose gel in sodium borate buffer at 225 V for 20 min. Bands were visualized under UV light (ChemiDoc MP system, BioRad, UK) after counterstaining with GelRed.

Storage Stability Study
The shelf-life stability of I-LPH siNEG-Eto was studied at 4 and −20°C for one month. The particle size, surface charge, and EE % of Eto and siNEG were determined as described above.

In Vivo Imaging and Organ Biodistribution
Animal experiments were conducted with project and personal licenses granted by the UK Home Office and in accordance with the UK Animals (Scientific Procedures) Act 1986 and UK Home Office Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (Home Office 1989). In vivo experimentation was adhered to the project licence approved by the King's College London animal welfare and ethical review body (AWERB). In imaging studies, three female BALB/c mice aged 4-6 weeks (Envigo, UK) were anesthetized using isoflurane and intravenously injected with 200 µL DiR-labeled I-LPH siNEG in PBS. Whole-body images of the anesthetized animals were captured at 0, 1, 4, and 24 h post-injection using the IVIS Lumina Series III In Vivo Imaging System (Perkin Elmer, USA) using excitation/emission wavelengths at 740/790 nm. Untreated animals were used as controls. Animals were sacrificed at 24 h post-injection and their vital organs (brain, heart, lung, liver, spleen, and kidneys) were collected, weighed, and imaged ex vivo.

In Vivo Therapeutic Study
Experimental mouse melanoma lung metastasis model was developed by injecting C57BL/6 mice (Envigo, UK) with 5 * 10 5 B16F10 cells suspended in 100 µL PBS intravenously via a tail vein. Animals were randomly divided into five groups (n = 7) namely: PBS, I-LPH siNEG , LPH siNEG-Eto , LPH siCD47 , LPH siCD47-Eto . Mice were injected intravenously with one of the above treatments on days 7 and 12 post tumor inoculation. PLGA, lecithin:tristearin, DSPE-PEG 2000, stearyl amine, Eto, and siCD47 doses were 50, 10, 10, 100, 20, and 0.1 mg Kg −1 respectively. Changes in body weight were recorded every 4 days. On day 23, animals were culled and major organs (lungs, heart, kidney, liver, and spleen) were collected and weighed. The success of the treatment was measured by comparison of lung weights between all the groups and more specifically counting the tumor nodules between LPH siNEG-Eto , and LPH siCD47-Eto .

Assessment of Immunological Changes in Lung Tissues
Immune cells were extracted from the lungs by physical maceration through a 70 µm cell strainer with a syringe. Extracted cells were stained with antibody cocktails including either anti-mouse CD8-PE and CD4-FITC or anti-mouse F480-FITC and CD11c-APC for 20 min. To establish a gating strategy, nonspecific isotype antibodies stained controls were also included. Cells were washed three times in PBS before being re-suspended in 200 µL of PBS. An equal quantity of precision count beads was added to each sample and run on a FACs BD FACSCalibur flow cytometer. Cells were analyzed using Flowjo software. Cells were first gated using forward/side scatter profile prior to the relevant marker being assessed, using isotype staining as background. For myeloid populations, cells were gated as described previously and relative levels of CD11c and F480 were analyzed in accordance with. [22] Using this strategy three cell populations could be broadly identified, and accordingly labeled: CD11c+, F480-dendritic cells, DC; CD11c+, F480+ alveolar macrophages, AM; CD11c−, F480+ interstitial macrophages, IM. The absolute number of each cell type was calculated by normalization of cell count to bead numbers and the cell numbers per lung weight were obtained by dividing the cell number by lung weight in mg. Data are presented as total cell number per mg of lung tissue.

Histological Examination
The collected organs including heart, lung, liver, spleen, and kidney from different treatments were fixed in 10 % neutral buffered formalin. Tissues were embedded in paraffin and sectioned for hematoxylin and eosin staining (H&E) according to the standard histological protocols at the Royal Veterinary College, UK. All stained sections were imaged using a Leica DM 1000 LED Microscope (Leica Microsystems, UK) coupled with a digital camera (QImaging, UK).

Statistical Analysis
Except for the therapy study, each experiment was conducted in triplicate in the same experiment and is a representation of two independent experiments, data were presented as mean ± SD (standard deviation). For therapy experiments, experiments were done once (n = 7), data were presented as mean ± SEM (standard error of the mean). Student's t-test was applied to compare two variables while ANOVA test followed by Tukey HSD test was used for comparing different parameters between groups. For lung nodules measurements, statistical analysis was carried out using student's T-test followed by Mann Whitney post-test. Differences were considered statistically significant at probability (p) value less than 0.05.

LPH Eto Formulation Optimization using BBD
Response surface methodology by using BBD was implemented to study the effect and the interactions of the different Adv. Healthcare Mater. 2021, 10,2001853 investigated CPPs on CQAs which were the particle size (Y1) and EE % (Y2). Table S2, Supporting Information, shows the composition of the fabricated 29 formulations and the obtained responses. The relation between different CPPs and each of the CQAs was expressed using a polynomial equation. The statistical models were selected based on the highest R 2 values (adjusted and predicted) with difference below 0.2 and the least PRESS value after removing the non-significant factors. [23] The signal to noise ratio, expressed as adequate precision, was greater than 4 proving the capability of models to navigate the design space. [17] The quadratic model was selected as the best fit statistical model (Tables S3 and S4, Supporting Information). The positive signs of the regression coefficient indicate a direct relationship between CPPs and CQAs, while the negative sign indicates an inverse relationship between them. [24] The 3D response surfaces are graphical illustrations, showing the relation between two CPPs while all other independent factors were kept constant.

Effect of Different CPPs on Particle Size (Y1) and EE % (Y2)
Tables S5 and S6, Supporting Information, listed the ANOVA of the effect of the investigated CPPs on particle size and Eto EE %, respectively, where P-values < 0.5 were considered significant. By inspecting Equation (5) and Figure S1, Supporting Information, a positive correlation between polymer, lipid, and drug concentration and PS could be observed. On the contrary, stirring speed is inversely proportional to particle size. The concomitant increase in both the polymer and drug had a positive effect on the particle size confirmed by the positive sign for AC in Equation (5) which is shown visually in Figure 1A. The interaction between lipid and drug however had an antagonistic effect on size demonstrated by the negative sign in Equation (5) and shown visually in Figure 1B. Equation (6) and Figure S2, Supporting Information, demonstrated that all the investigated parameters showed a positive influence on Eto EE %. Figure 1C showed a negative influence of the interaction between the polymer and the lipid on EE %. Design space is multidimensional CPPs combinations and their interactions, identified through design of experiments data and had been verified to afford QTPP. [24] The design space is plotted by overlapping different CPPs' influences on CQAs contour plots to obtain QTPP. The yellow area represents the values of CPPs when optimized to fulfill QTPP criteria; minimum particle size and maximum EE % ( Figure S3, Supporting Information).
Based on the high desirability, one formula was selected and prepared as a checkpoint to validate the developed models. Scheme 1 and Table S7, Supporting Information, illustrate its composition and the corresponding predicted and experimental particle sizes and EE % values respectively. The percentage error was <4 % thus the developed model was judged suitable for studying and predicting CPPs for the preparation of LPH Eto with the desired QTPP. This formula was used for the incorporation of siRNA using either preparation methods, in situ (I pre-fix) or post-incorporation of siRNA (P pre-fix), with or without Eto. Table 1 shows that all P-LPH formulations had a significantly higher particle size than their respective I-LPH type (P < 0.05). All the prepared LPH had PDI < 0.2 confirming the monodispersity of the system. All the proposed formulae had a positive surface charge which significantly diminished upon siRNA incorporation (P < 0.05). Eto EE % ranged from 74.67 ± 5.35 to 81.59 ± 2.55 % which was not affected by the presence of siRNA. The LE % of Eto and siRNA ranged between 6-8 % and 0.1-0.3 nmole siRNA/mg LPH, respectively. The obtained LE results are in accordance with previously reported studies. [21] The presence of siNEG had insignificant effect on Eto LE%. On the other hand, Eto significantly reduced siNEG LE % (P < 0.05). I-LPH with or without Eto had significantly higher LE than P-LPH (P < 0.05). The LPH retained their starting size, surface charge, drug, or siRNA EE % (Table S8, Supporting Information). Transmission electron microscope (TEM) images of I-LPH siNEG-Eto revealed the presence of spherical-shaped, non-aggregated nanoparticles in the size range of ≈100-110 nm, consistent with the DLS measurements (Figure 2A). The coreshell structure could be visualized in the photomicrographs; the white core and dark zone represent the polymeric matrix and the lipid coat, respectively, confirming the core-shell structure of LPH. [25]

LPH Exhibits Controlled Drug Release Characteristics and Reduces Blood Hemolysis of Eto
The optimized LPH formulation demonstrated a biphasic release profile which began to plateau from 12-24h. The release rate was approximately 6.3 % h −1 in the first 8 h followed by a more controlled rate of approximately 2.2 % h −1 in the second phase (from 8-24 h). A total of 20 %, 50 %, and 80 % releas was measured at 2, 8, and 24 h, respectively, in the presence of 50 % FBS at pH 7.4 compared to a 100 % release achieved within 3 h in the free form of the drug ( Figure 2B).
The hemolytic activity of LPH Eto was compared to that of the free drug ( Figure 2C). Eto solution showed a dose-dependent hemolytic activity with hemoglobin release ranging from 12.6 to 62 %, in agreement with previous reports. [26] On the contrary, less than 5 % of hemoglobin release was noted for LPH at equivalent Eto concentrations at a maximum of 100 µg mL −1 , which is within the acceptable range of the new consensus ASTM E2524-08-Standard test method for analysis of hemolytic properties of NPs. [27]

LPH Formulation Shows Increased Anti-Cancer Activity Compared to Free Drug and can be Used to Deliver siRNA
A dose-dependent reduction in B16F10 cell viability was observed after incubation with Eto solution or LPH Eto at a concentration range of 0.01-100 µM after 48 h incubation ( Figure 2D). The IC 50 for Eto solution or LPH Eto was 0.6611 and 0.1723 µM, respectively. In addition, the calculated IC 50 of I-LPH siNEG-Eto and P-LPH siNEG-Eto was 0.1791 and 0.1893 µM respectively ( Figure S4A, Supporting Information). Drug-free LPH with or without siNEG exhibited > 80 % cell viability up to 10 µM equivalent drug concentration ( Figure S4B, Supporting Information). A significant reduction in cell viability at higher concentrations could be at-tributed to the presence of stearyl amine. Altogether the results cooperated that the enhanced toxicity of Eto in an LPH form is not due to an additive toxicity of the nanocarrier.
The ability of I-LPH and P-LPH to improve the cellular internalization of fluorescently labeled siRNA was assessed in B16F10 cells at two concentrations (30 and 90 nM) and two incubation periods (4 and 24 h) with/without Eto (0.1 µM). The extent of cellular uptake was expressed as MFI. Figure 3A shows a representative histogram of Atto655-siRNA cellular uptake. Both I-LPH and P-LPH (p > 0.05) improved siRNA uptake in a time-dependent fashion ( Figure 3B). A significant reduction in CD47 expression by 50-60 % was observed independent of LPH types and doses ( Figure 3C-D). No gene silencing was    obtained with LPH siNeg (Figure S5, Supporting Information) suggesting that silencing is specific and is not a toxicity artefact. Additionally, Eto had an insignificant effect on siRNA internalization and siCD47 silencing (p > 0.05) ( Figure S6, Supporting Information).

LPH are Stable in the Presence of Serum
Changes in size, PDI, and zeta potential of LPH were assessed following incubation with 50 % serum for 4, 24, and 48 h (Figure 4A-C). In contrast to P-LPH, I-LPH maintained the size, PDI, and zeta potential up to 24 h incubation. A significant increase in size and PDI and a reduction in the zeta potential of both types were observed after a 48-h incubation (p < 0.05). The ability of LPH to protect the entrapped siRNA from serum and RNAse degradation was tested ( Figure 4D). Expectedly, naked siRNA suffered from extensive degradation. I-LPH offered better siRNA protection from FBS and RNAse than P-LPH, evident by the comparable band intensities to untreated siRNA. Due to the overall better stability and higher siRNA EE % of I-LPH compared to P-LPH , the former was selected for further in vivo evaluation.

DiR-LPH Accumulates in MPS Organs of Mice after Intravenous Injection
The biodistribution profile of DiR-labeled I-LPH siNEG was studied over 24 h after a single intravenous injection. Figure 5A shows the whole-body images of mice taken at 0, 1, 4, and 24 h postinjection. High accumulation of LPH in liver was captured up to 24 h, indicating the retention of LPH in liver. Uptake in spleen was also shown by whole-body imaging. No signs of clearance were observed at 24 h, the latest studied time point in this study. Ex vivo images of major organs confirmed the level of accumulation of LPH in the major MPS organs in the order of liver, spleen, and lung ( Figure 5B,C). This imaging study suggested the candidacy of LPH to deliver Eto/siRNA to metastatic cancer to lung or the liver.

Dual siCD47 and Eto Therapy Resulted in Reduced Tumor Nodules in the Lungs Compared to Monotherapy
The therapeutic efficacy of I-LPH siCD47-Eto was evaluated in the experimental lung metastatic B16F10 tumor model after two . I-LPH siNEG shows a superior protective effect of the encapsulated nucleic acid against serum and RNAse. The selected Eto siNEG are incubated with PBS and 50% FBS for 4, 24, and 48 h then A) particle size, B) PDI, and C) zeta potential are measured using DLS as described. Data points represent mean and SD (n = 3). Statistical analysis is carried out using student's t-test *p < 0.05. The stability of the encapsulated siRNA is assessed by incubation with 50% v/v FBS or RNAse (100 µg mL −1 ) for 48 h. RNAse activity is inhibited by addition of EDTA before treating LPH with heparin (100 IU). Released RNA is qualitatively assessed using gel electrophoresis. siRNA (untreated) and naked siRNA are used as a negative and positive control, respectively (D).
intravenous injections. Monotherapies with siCD47 and Eto in LPH were tested for comparison. Mice in both PBS and I-LPH siNEG groups showed significant weight loss (p< 0.05) compared to monotherapy (I-LPH siNEG-Eto and I-LPH siCD47 ) and dual therapy group (I-LPH siCD47-Eto ) suggesting increased disease severity (Figure 6A). Post-mortem macroscopic analysis (day 13) demonstrated apparent reduction in melanoma nodules in the following order: I-LPH siCD47-Eto > I-LPH siNEG-Eto > I-LPH siCD47 > I-LPH siNEG and PBS groups ( Figure 6B and Figure S7, Supporting Information). Significantly lower lung weights were observed in both Eto treated groups (I-LPH siNEG-Eto and I-LPH siCD47-Eto ) (p < 0.05) in comparison with the other groups ( Figure 6C). Lung weight values for I-LPH siCD47-Eto, I-LPH siNEG-Eto, I-LPH siCD47, I-LPH siNEG , and PBS were 0.237 ± 0.087, 0.326 ± 0.099, 0.65 ± 0.14, 1.024 ±0.063, and 0.832±0.143 g, respectively. In addition to lung weight measurements, we employed another method to quantify tumor burden by counting the tumor nodules in the lungs ( Figure 6D). Nodules numbers were counted in the drug monotherapy and combinatory therapy groups only since it was difficult to find well-defined isolated nodules in the control, siCD47/siNEG monotherapy groups due to excessive tu-mor growth. Nodule counts confirmed that combinatory therapy is significantly more effective than drug monotherapy in reducing tumor burden, which agreed with macroscopic analysis (Figure 6B) and histopathological analysis ( Figure S8, Supporting Information).

Dual siCD47 and Eto Therapy Resulted in Altered Lung Leukocyte Population Density in the Lungs
To establish if there was a correlation between the anti-tumor activity observed and immune responses, we performed immunological assessments in tumor-bearing lung tissues. It was observed that all mice receiving Eto containing I-LPH had significantly elevated numbers of CD8+ cells when compared to mice receiving the I-LPH siNEG (Figure 7A). This increase was most pronounced in the case of mice receiving I-LPH siCD47-Eto however, significance between I-LPH siCD47-Eto and I-LPH siNEG-Eto could not be obtained, possibly due to variation between mice. This general trend was conserved in the CD4+ population however, in contrast, levels in the I-LPH siCD47-Eto group were significantly higher than the I-LPH siNEG-Eto group ( Figure 7B) . Due to the proposed mechanism of siCD47, we were particularly interested in the lung myeloid cells, especially antigen-presenting cells. To address this, we utilized a relatively simple staining strategy to identify three putative cell populations, these being: IM, AM, and DC ( Figure 7C, 7D, and 7F respectively). A representative flow cytometry dot plot is shown in Figure 7E. Using this analysis, it was observed that there were the highest number of IMs present in mice receiving I-LPH siCD47-Eto , this being the only group significantly different from I-LPH siNeg ( Figure 7C) . Interestingly, there were elevated levels of AMs in mice treated with I-LPH siCD47 and I-LPH siCD47-Eto. These differences were significant compared to I-LPH siNeg but not to I-LPH siNeg-Eto. I-LPH siNeg-Eto , in turn, was not significantly different from I-LPH siNeg ( Figure 7D). The DC numbers followed no discernible trend and there were no significant differences observed between any of the treatment groups (Figure 7F).

Histological Analysis
Microscopic examination on lung tissues further confirmed the therapeutic effectiveness of LPH containing Eto, siCD47, or the combination ( Figure S8, Supporting Information). Large areas of tumor invasion were observed in lung tissues of the PBS and I-LPH siNEG groups, whilst more areas of alveoli and bronchioles were present in the lungs of mice treated with I-LPH siNeg-Eto or I-LPH siCD47 . In the I-LPH siCD47-Eto group, most of the lung tissues showed normal histological features and only a few small tumor nodules were observed. Histological examination was also carried out on major organs including heart, liver, spleen, and kidney. As shown in Figure S9, Supporting Information, no obvious histologic changes were observed in these organs in the treatment groups compared to the PBS group.

Discussion
Many tumors suppress the host immune system allowing for immune escape and leading to tumor growth and progression. [28][29][30] Various types of tumors can circumvent the host immune clearance by overexpression of CD47. [31] Targeting CD47 using monoclonal antibodies or siRNA has been suggested previously. [2,3] Indeed, there is data to suggest this is a viable approach both preclinically and clinically. [32][33][34] However, one future drawback of the use of CD47 based therapeutics may be that it is relatively widely distributed, including on RBCs. [35] Administration of anti CD47 monoclonal antibodies intravenously may result in rapid dilution and toxicity through off-target effects. [36] The use of In (A-C), statistical analysis is performed using one-way ANOVA followed by Tukey post-test *p < 0.05. Bars represent mean ± SEM. In (D), statistical analysis is carried out using student's t-test followed by Mann Whitney post-test *p < 0.05.
siCD47 formulated in a particulate system could possibly negate or minimize these issues by blocking the CD47 axis at a more local level. As CD47 is proposed to exert its effects through the phagocytosis activity of macrophage, it may be speculated that CD47 therapy is most suited to tumors or organs with high levels of macrophages, either tumor-associated or tissue-resident. [37] To this end, we designed an LPH for delivery to the lung. As there is an abundance of macrophage present in the lung, targeting them, via the CD47 axis, rather than T cells via the PD-1, CTLA4 axis may have a logical foundation. [38] To compliment lung delivery of siCD47 etoposide was selected. Eto is currently approved in clinic as part of the lung cancer chemotherapeutic regimen and is effective against the B16F10 cell line used in the pseudo metastatic mouse model and therefore a viable candidate. [39] Generally, the physicochemical properties such as size of nanocarriers dictate their intracellular internalization and their subsequent therapeutic applications. [40] In addition, higher drug EE % is an important factor in the clinical translation of different nanoparticles. In an attempt to produce LPH with an optimal size <150 nm and maximum Eto EE %, different formulation parameters and processes were employed and the effects on size and EE % was studied. The concomitant increase in particle size with increasing polymer, lipid, or Eto concentrations could be attributed to the increase in viscosity which inversely affects the evaporation rate of the organic solvent and opposes the effect of stirring speed to breakdown the particles into smaller ones. [41][42][43] On the contrary, increasing the stirring speed increases the mechanical and hydraulic shear which decreases the particle size. [44] PLGA, lecithin:tristearin, and Eto concentration, as well as, stirring speed showed a positive effect on EE %. The pronounced effect of increasing PLGA concentration on augmenting EE % was attributed to the availability of larger core material to Data is plotted as mean ± SEM and is analyzed with Graphpad Prism 8 using students t-test with Mann Whitney post-test ns, non-significant *p < 0.05, **p < 0.005. incorporate drug. [45] Additionally, the higher organic phase viscosity and larger particle size accompanying higher polymer amounts likely prevented the formation of porous particles with the procurement of longer diffusion path for the drug, enhancing its encapsulation. [46,47] Increasing lipid concentration also improved the EE % likely by forming a physical barrier that counteracts Eto diffusion from the core. [48] Increasing the stirring speed favored higher EE % likely by increasing the solubility of Eto into the formed polymeric core during the nanoprecipitation process.
All the prepared siRNA-Eto LPH had a particle size of less than 150 nm to take advantage of accumulation into tumor via the EPR effect. [49] siRNA EE % was twofold higher for I-LPH than P-LPH (p < 0.05) due to the difference in the incorporation method. A sustained drug release profile is a key factor circumventing the undesirable leakage into circulation and consequently aids in achieving the desired therapeutic effects. [50] LPH which incorporates the features of both polymer and lipid nanocarriers could control the Eto release for 24 h. The presence of lipid shell prevents drug diffusion and could hinder water penetration into the polymeric core that could reduce the degradation rate of the PLGA core. [12] The biphasic release is well described for this type of formulation, [51] the initial release is due to drug being lost from the surface of the particle. This burst release is followed by slower release rate due to the partitioning of the drug from the core to the release medium.
The incorporation of Eto into LPH effectively improved its in vitro cytotoxicity against B16F10 cells by 3.8-fold compared to free drug. The improvement of potential Eto cytotoxicity after encapsulation in nanoparticles was previously reported in different types of nanocarriers as liposomes, [52] PLGA nanoparticles, [53] albumin nanoparticles, [54] and poly (N-isopropylacrylamide) coated superparamagnetic iron oxide nanoparticles. [55] The ability of nanoparticles to improve cytotoxicity is not exclusive for Eto but was also reported for several other anticancer drugs as paclitaxel, [56] cisplatin, [57] Epirubicin, [58] and doxorubicin. [59] This could be attributed to the fact that about 50 % of the anticancer drugs, including etoposide, are substrates to P glycoprotein (Pgp) mediated efflux. [60] Nanoparticles are an efficient tool to circumvent p-gp. [61] In addition, Tween 80 incorporated in the LPH formulation has been previously reported as P-gp inhibitor. [62] High siRNA cellular uptake is a prerequisite for successful gene silencing efficiency. By virtue of their high molecular weight and condensed polyanionic structure, naked siRNA could not cross the cell membrane. On the contrary, LPH was able to improve siRNA cellular internalization as indicated by higher MFI values. The higher siRNA uptake consequently correlated with the successful downregulation of CD47 expression (≈50 %). Using LPH, 50 % knockdown of CD47 was achieved using siRNA in the range of 30-100 nM in a dose-independent manner, this level of knock down is consistent with previous reports, [63] however greater knock down was achieved by Wang and co-workers who used a liposome-protamine-hyaluronic acidbased system. [64] This suggests that knockdown efficacy may be limited by the delivery vector or cell type. The significantly higher stability of I-LPH over P-LPH could be due to the siRNA encapsulation method with the former offering better protection against siRNA desorption and serum attack. [12] A dose-dependent effect on CD47 silencing could not be observed in our study. Although surprising, similar results were obtained by Thanki et al, where 2′-O-methyl-modified dicer substrate asymmetric siRNA duplexes, directed against enhanced green fluorescent protein (EGFP-siRNA) loaded LPH, showed dose-dependent gene silencing at concentration range 1-10 nM with values plateauing at concentration range of 10-100 nM in H1299 mEGFP cells. [65] In accordance with the common feature reported for many nanoparticles, high amounts of LPH were detected in the liver, spleen, and lung, the major MPS organs. Although it is difficult to discriminate lung cells from tumor cells by whole body optical imaging due to the resolution of this technique, the significantly improved antitumor efficacy obtained after the systemic administration of I-LPHsiCD47-Eto is likely to be attributed to the effective delivery of LPH to the cancerous nodules. Despite delivery to both healthy lung tissues and cancerous nodules, it is known that chemotherapeutic drugs and RNAi have more pronounced effects in rapidly dividing cells (tumor) compared to non-dividing cells (healthy lung tissues) which could have led to the selective pharmacological toxicity observed. This hypothesis was supported by the normal histological appearances in liver and spleen in the mice which received I-LPH siNEG or drug-loaded LPH treatments.
High amounts of LPH were detected in the lung. The significantly improved antitumor efficacy obtained after the systemic administration of I-LPH siCD47-Eto could be attributed to the effective delivery of LPH to the lung, leading to the release of the cytotoxic effect of Eto and the successful transfection of siCD47. This observation demonstrates hypothetical synergistic or additive effects of combining both Eto and siCD47. We have demonstrated that macrophage numbers (innate activation) and T cell numbers increased (innate/adaptive activation) suggesting that treatment with siCD47 LPH activates the immune responses. It is widely reported that macrophage can phagocytose cancer cells and this effect is improved with CD47 blockade. [66] Indeed, this has been previously shown in the B16F10 lung metastatic model using siCD47. [64] As shown in the data the numbers of T cells remain relatively low, so it was not possible to extract enough numbers of T cells from lungs to perform ex vivo activation studies. We however speculate that the increase in T cell numbers is a direct response to macrophage activation (release of cytokines, establishment of chemokine gradients, etc.).
Eto itself has a complex interplay with the immune system depending on the model and the criteria used for assessment. It has been shown that splenocytes adoptively transferred from mice which resolved tumor following Eto treatment can protect naïve mice from tumor challenge. [67] This strongly suggests that antitumor immunity is established successfully. The authors speculate that this may be due to the drug's effects on the tumor cells, causing the release of antigen containing apoptotic bodies to antigen-presenting cells, including macrophages. Interestingly, it has also been reported that Eto selectively ablates activated T cells, in a hemophagocytic lymphohistiocytosis model leading to immune suppression. [7] In the same study, it was noted that following in vivo Eto treatment macrophages maintain the ability to present antigen and mature. [7] The detailed role of Eto in cancer treatment and immune system modulation may seem elusive but studies have suggested that cancerous cells killed through Eto treatment induce macrophages to produce TNF and other pro-inflammatory signals unlike some other chemotherapeutic agents. [68,69] Although, Eto is not considered to be a classic "im-munogenic cell death" inducer as it does not induce translocation of calreticulin, it can however cause the release of inflammatory mediators ATP and HMGB1. [70] Conversely, macrophages have been shown to either enhance or reduce Eto-induced apoptosis depending on the phenotype (i.e., M1 or M2 macrophages). [71] In our study, although the activation state of T cells was not assessed, we observed elevated numbers of T cells in the lungs with I-LPH siCD47-Eto treatment. It is possible that, as the lung has a distinctively high macrophage population, the proinflammatory activity of the macrophages on the T cells predominates. It could be speculated that following the combinatory treatment, Eto provides a source of apoptotic tumor cell fragments which can provoke an inflammatory response. These fragments can be taken up to a greater extent by antigen-presenting cells including macrophages, as a result of concomitant CD47 axis blockade.
In this study, we were able to demonstrate that the co-delivery of siCD74 and Eto in a single system could both reduce tumor growth and induce both innate and adaptive immune responses. It will be interesting to test in future studies whether using a subtherapeutic Eto dose combined with siCD47 for example will show a similar therapeutic effect to the therapeutic Eto treatment as a monotherapy. Additionally, this study showed that the co-delivery of a chemotherapeutic drug such as Eto, as a model drug, and a siRNA in the LPH system developed is therapeutically useful so future studies will focus on replacing Eto with other chemotherapeutic drugs with known immunomodulatory activities such as doxorubicin at subtherapeutic and therapeutic doses. [72]

Conclusion
The current study confirmed the feasibility of the proposed LPH system as a platform for the simultaneous delivery of Eto and siCD47 for the treatment of lung's metastasized tumor nodules. The combinatory treatment of Eto with siCD47 delivered by the preferred LPH formulation offered an improved therapeutic outcome compared to drug monotherapy in the lung metastatic model of melanoma B16-F10 cells. The anti-tumor responses correlated well with immune cell activation pattern in the tumorbearing lungs.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.