Development of a concentration‐controlled sequential nanoprecipitation for making lipid nanoparticles with high drug loading

Lipid‐based nanostructures have garnered considerable interests over the last two decades, and have achieved tremendous clinical success including the first clinical approval of a liposome (Doxil) for cancer therapy in 1995 and the recent COVID‐19 mRNA lipid nanoparticle vaccines. Compared to liposomes which have a lipid bilayer surrounding an aqueous core, lipid nanoparticles with a particle structure have several attractive advantages for encapsulating poorly water‐soluble drugs such as better stability due to the particle structure, high drug encapsulation efficiency because of a pre‐ or co‐drug‐loading strategy. While many studies have reported the synthesis of lipid nanoparticles for hydrophobic drug encapsulation, the precise control of drug loading and encapsulation efficiency remains a significant challenge. This work reports a new concentration‐controlled nanoprecipitation platform technology for fabricating lipid nanoparticles with tunable drug loading up to 70 wt%. This method is applicable for encapsulating a wide range of drugs from very hydrophobic to slightly hydrophilic. Using this facile method, nanoparticles with tunable drug loading exhibited excellent properties such as small particle size, narrow size distribution, good particle stability, showing great promise for future drug delivery applications.


INTRODUCTION
Lipid nanoparticles (LNPs) have emerged as promising therapeutic delivery vehicles for a wide range of drugs over the past few decades.The amphipathic and biodegradable nature of the lipid nanomaterials makes them attractive for encapsulating both hydrophobic and hydrophilic therapeutic drugs for drug delivery. [1]The great success of COVID-19 mRNAlipid vaccines have rightly put lipids on the spotlight again. [2]NPs can be categorized into two main groups: liposomes with a water core and lipid bilayer structure, and solid LNPs.However, most LNPs in the clinical context have limited drug loading (< 10 wt%).[3] While the emulsion/solvent evaporation method can produce NPs with drug loading up to 14%, other common approaches such as traditional nanoprecipitation method or evaporation method can only achieve a drug loading less than 5%.[4,5] Such a low drug loading requires the excessive use of nanocarriers to achieve the therapeutic threshold of drugs, leading to toxic side effects and degradation burden.[6] In contrast, high drug loading NPs offer advantages such as improving intravenous injection compatibility, minimizing material toxicity, reducing manufacturing cost and increasing production scalability.[3,6] Meanwhile, it has been demonstrated that cells are only able to uptake a limited number of NPs.According to a previous research, SKOV3 cells have been shown to have a limited capacity for NPs cellular uptake, with a maximum of 1.9 × 10 5 particles taken up by a single SKOV3 cell.[7] Therefore, a minimum number of NPs with sufficient amount of drug loaded is beneficial for delivering a maximum amount of drug. [7]It is therefore of great significance to develop LNPs with tunable drug loading, ranging from low to very high, thereby reducing the need for excessive use of nanocarriers and improving drug delivery efficacy.
14] Among the approaches, nanoprecipitation is the simplest method for lipid-based nanoparticle production, and does not require high-shear force/high-temperature/high-pressure. [15]riefly, nanoprecipitation employs two miscible solvents, one (the organic phase) containing drug and lipids, and the other containing the antisolvent (the aqueous phase). [16,17]he NP forms quickly upon mixing as the drug containing organic solvent diffuses into the aqueous phase, leading to solvent displacement. [16,18]However, NPs produced with this method often have a low drug loading (less than 10 wt%) due to the significant difference in the precipitation time between the drug and lipid materials.The quicker precipitation of lipid materials than the drug leads to drug aggregation or crystallization, resulting in only a small amount of drug being encapsulated within the NPs. [3,19]Alternatively, the sequential nanoprecipitation technology developed in our laboratory enables the precipitation of the drug first followed by the polymers by adjusting the volume ratio of multiple solvents in the organic phase, thus forming drug-core polymer-shell NPs with tunable drug loading from low to high. [20,21]However, this sequential nanoprecipitation has not been explored for making lipid-based NPs with tunable drug loading.
Liposomes are one of the most common subtypes of lipid-based NPs for drug delivery. [22]Over the years, many successful cases of drug-loaded liposomes have been developed for clinical applications, such as Doxil, Myocet, Vyxeos, etc. [23] Depending on their physicochemical properties, water-soluble therapeutics are predominantly encapsulated within the aqueous core of the liposomes, whereas lipophilic drugs are incorporated into their phospholipid bilayer matrix. [24]However, despite their well-established fabrication method and applications, liposomes as a drug delivery carrier suffer some limitations, especially for delivering hydrophobic therapeutics.The low solubility of these therapeutics in aqueous medium makes it difficult to load sufficient active therapeutics into the liposomes.Additionally, the nature of the lipid bilayer structure of liposomes can lead to easy leakage, resulting in only a very small amount of cargos reaching the intended target. [25]erein, we report a facile and efficient concentrationcontrolled nanoprecipitation method for synthesizing LNPs with high drug loading up to 70 wt% and encapsulation efficiency (EE%) higher than 85% as well as good stability.The hydrodynamic properties of the LNPs, including particle size, polydispersity, and zeta potential, were characterized, their morphologies were examined using Transmission Electron Microscope (TEM), and their stability, drug loading capacity (DL%) and EE% were determined.This work introduces a new library of lipid-based nanoparticles with exceptional properties, representing a significant advancement in lipid-based nanoparticle technology.

Screening and preparing exceptionally high drug loading lipid-based NPs
To determine the optimal formulation for LNPs with high drug loading, a simple concentration-controlled sequential nanoprecipitation method was developed to prepare LNPs with exceptionally high drug loading at room temperature.In general, single or dual hydrophobic drugs (DTX, DOX, CCM, CPT, ibuprofen) and single or multi-lipids (DMPC and cholesterol with different mass ratio or DMPC only) were dissolved in single or multi-component organic solvent which was composed of acetonitrile, methanol, Ethanol (EtOH), Dimethyl sulfoxide (DMSO) or Dimethylformamide (DMF) at a concentration ranging from 1 mg mL −1 to 30 mg mL −1 in a glass vial as an organic precursor.Specifically, DSPE-PEG (2 kDa) was dissolved in an organic phase at 5 mg mL −1 for the PEGylated samples.Subsequently an aqueous solution (water, phosphate buffered saline 1 × PBS which were adjusted to neutral pH or HEPES which was adjusted to pH ∼ 7.5) was rapidly added to the organic precursor containing drug and lipids with around 19 times volume of the organic phase, and then mixed under magnetic stirring with a speed of 550 revolutions per minute (RPM).Then this nanosuspension was dialyzed with a 10 KDa molecular weight cut off dialysis membrane (Merck Millipore, Germany) against water or PBS buffer or HEPES buffer for at least 12 h in 4 • C cold room or at least 2 h at room temperature to remove the residual solvents.After the preparation, the samples were collected for further analysis.For those samples having sizes around 100 nm and polydispersity index (PDI) values equal or less than 0.2, the corresponding formulations were determined as good formulations for future characterization.

Characterization of lipid-based NPs with high drug loading
The size, PDI and zeta potential of LNPs with high drug loading were characterized using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) at 25 • C. The particle size stated in the paper is hydrodynamic particle size measured by DLS, unless otherwise stated.The morphology and dry size of lipid-based NPs were observed using a Hitachi HT7700 TEM (Hitachi Ltd., Tokyo, Japan) or a FEI Tecnai G2 Spirit BioTwin TEM machine (Oregon, USA).To prepare the samples, 10 µL of bulk samples were dropped onto a formvar-coating copper grid.After 3-min air-drying at room temperature, the redundant solution left on the grid was absorbed by a filter paper gently followed by 3-min negativestaining using 2% uranyl acetate (UA) and absorbed by a filter paper again.

Characterization of drug loading capacity and encapsulation efficiency
To determine DL% and EE%, freshly prepared samples were left undisturbed at room temperature for 2 h with aluminium foil covering to allow for complete aggregation of free drug, as the aggregates may result from recrystallization of unencapsulated free drugs or drug leakage from unstable NPs.Then the bulk samples were centrifuged at a slow speed of 500 × g for 1 min to remove any large drug aggregates.Next, 800 µL of supernatant was frozen at −80 • C, covered by aluminium foils for at least 1 h and lyophilized at least for 24 h until dry crystalline was obtained.Then freeze-dried samples were fully dissolved by adding proper solvent and vortexed for 5 mins.The drug concentration in the dissolved samples was determined using a reversed-phase high performance liquid chromatography (RP-HPLC) (Shimadzu Corporation, Kyoto, Japan) or a UV-vis spectroscopy (Shimadzu Corporation, Kyoto, Japan).The standard curves of different drugs in solvents are shown in supporting information (Figure S1).
Drug-loaded LNPs EE% was defined in Equation (1

Stability studies
The freshly-prepared samples were stored in 4 mL glass vials with caps, and covered with aluminium foil to maintain their integrity, at room temperature.At predetermined time points (0, 24, 48, 72 h, etc.), the hydrodynamic properties of samples, such as size and PDI values were determined using Dynamic Light Scattering (DLS).Concurrently, the status of the bulk solutions was monitored by observation.

Precipitation titration curve
To obtain the precipitation curve of drugs and materials in situ, a 400-µL solution of drug-DMSO (5 mg mL −1 ) or lipid-ethanol (5 mg mL −1 ) was prepared, and 80 µL of antisolvent (aqueous) was rapidly added with pipette mixing.Then, the derived count rates (DCR) values were measured using DLS, and the results were recorded as N for each subsequent addition of 80 µL or 40 µL of antisolvent to the drug or lipid-containing solution.For example, when 160-µL water (i.e., water/solvent volume ratio = 0.4:1) was added to the DTX-DMSO solution, the DTX started to precipitate rapidly (around 75% precipitated out).The minimum water/solvent ratio was 1:1 (v/v) to ensure most of the DTX (5 mg mL −1 ) precipitated.The total amount of nanoparticles obtained when they fully precipitated out (DCR × volume) was denoted as Nt.The precipitation titration curves were generated by plotting the volume of antisolvent/volume of solvent ratio against the cumulative precipitate percentage (X axis: volume of antisolvent /volume of solvent ratio; Y axis: cumulative precipitate% = N / Nt * 100).These curves were generated for DTX-DMSO (5 mg mL −1 ) solutions with different concentrations of lipids.

In vitro drug release study from lipid-based NPs
The in vitro drug release study was performed under sink conditions.The solubility of DTX was around 8 µg/mL in phosphate-buffered saline (PBS; pH 7.4), and increased to 38 µg/mL when dissolved in 0.2% (w/v) Tween 80 PBS buffer.200 µL of DTX-loaded LNPs suspension were sealed in dialysis membrane with a molecular weight cut-off (MWCO) of 10 KDa and dialyzed against 1.8 mL PBS buffer containing 0.2% (w/v) Tween 80 for 12 h.In this case, the volume of release medium was higher than three times of the saturation volume of DTX sealed within the dialysis membrane, thus this experiment meets the requirements of sink conditions defined in European Pharmacopoeia.The in vitro release study was conducted in an incubator with a mild shaking speed of 150 rpm and a temperature of 37 • C ± 0.5 • C, covered by aluminium foils.Note that 100 µL of release medium was withdrawn at 0.5, 1, 2, 4, 8, 12 h and 5.26-µL DMSO was added and mixed for analysis, and replaced with the same volume of fresh medium to maintain the sink condition in the system.All the experiments were carried out in triplicate.The samples were analyzed using HPLC.The cumulative drug release (CDR) % was calculated as: CDR% = Mass of drugs at specific time + Total Mass of drugs withdrawn before this specific time Actual drug input mass × 100%

RESULTS AND DISCUSSION
Herein, we report a facile nanoprecipitation technology for preparing drug-loaded LNPs with tunable drug loading from low to exceptionally high (up to 70 wt%).In contrast to traditional liposomes that utilize a remote loading approach to load a drug, our technology achieves nanoprecipitation by controlling the precipitation time of drug and lipids, ensuring that hydrophobic drugs precipitate first, followed by the formation of a lipid shell, resulting in a drug-core lipid-shell NP with high drug loading (Figure 1).Additionally, our results demonstrate that this method is effective not only for highly hydrophobic drugs such as DTX, CCM, CPT, but also for slightly hydrophilic drugs such as DOX, Ibuprofen (Table S1).
Previously, we used solvent mixtures with three different solvents to tune the precipitation time of the drug and polymer, [20] as the precipitation of polymer is very sensitive to solvents, but not to its concentration.In contrast to polymer systems, the precipitation of lipids also depends on their concentration in addition to the solvent system and vol-ume ratios.Therefore, we develop a concentration-controlled sequential nanoprecipitation method that allows for the tuning of drug and lipid precipitation times, resulting in the sequential formation of a drug core followed by the lipid shell.Figure 2 shows the precipitation titration curve of docetaxel (DTX) and lipids.With a higher drug-to-lipid mass ratio, DTX precipitates faster than the lipids thus forming drug-core lipid-shell NPs.When the drug concentration decreases and lipids concentration increases, the precipitation curves of them move closer until they almost overlap.This concentration-controlled sequential nanoprecipitation can be applied to a wide range of drug-lipid systems.Using this method, we developed DTX-loading, CCM-loading, CPTloading, ibuprofen-loading, and DOX-loading LNPs with optimal drug loading (Table S1), which we will discuss in more detail in the following sections.

Synthesis and characterization of high DTX-loading LNPs
Docetaxel (DTX) is selected as a model drug as it is hydrophobic and has been widely used for cancer treatment.To investigate their capability in forming nanoparticles, free DTX and pure lipids were firstly precipitated.Free DTX cannot form NPs, as pure DTX aggregated rapidly after being produced using nanoprecipitation with a very large size (1726 ± 608 nm).Pure LNPs were synthesized using pure DMPC, pure cholesterol, DMPC/cholesterol (DMPC/CHO) at mass ratios of 2:1 and 4:1, respectively.Both pure DMPC and DMPC/CHO formed LNPs with low PDI (<0.2), but the size of pure DMPC NPs was much smaller (46.5 ± 0.2 nm) than the DMPC/CHO NPs (116 ± 2 nm and 97 ± 2 nm) (Figure 3A).In contrast, pure cholesterol generated larger NPs with a wider size distribution (PDI = 0.4).
To make DTX-loaded LNPs, a single-component lipid (DMPC/CHO 1:0) could not form good NPs (size = 217 ± 5 nm; PDI = 0.5) (Figure 3A; Table S2).In contrast, when cholesterol was used, the DTX-loaded NPs (DMPC/CHO 0:1) with high drug loading were produced with a PDI of 0.17, highlighting the crucial role of cholesterol in stabilising DTX.Following the initial screening study, two optimal formulations were identified for making LNPs with high DTX loading (40%DL), that is, LNPs containing DMPC and cholesterol at mass ratios of 2:1 and 4:1.They both exhibited smaller particle sizes (134 ± 2 nm and 157 ± 1 nm, respectively) and a narrow size distribution (PDI = 0.03 and 0.04, respectively).Although DTX-loaded LNPs with 50%, 60% even 70% DL have been successfully fabricated with good size and PDI values, aggregates were observed within 72 h after production at room temperature.Therefore, LNPs with 40% DTX drug loading were chosen for further studies.
PEGylation is a critical step in preventing nonspecific binding and reducing particle aggregation. [26]Therefore, PEGylation would offer benefits for the application of high drug loading LNPs.To prepare PEGylated DTX-loaded LNPs (40%DL), the two formulations with mass ratios of DMPC to cholesterol at 2:1 or 4:1 were selected for making NPs with different PEG content and synthesis conditions.Given the lack of prior research on high drug loading LNPs, DSPE-PEG 2 kDa at a concentration of 1 wt% PEG (W PEG / (W drug + W lipid )) was initially studied with different synthesis conditions (water, PBS and HEPES) to investigate whether PEGylation affects the hydrodynamic properties of LNPs (Table S2).Our results showed that all the 1 wt% PEGylated DMPC/CHO 2:1 NPs and 1 wt% PEGylated DMPC/CHO 4:1 NPs displayed good size distribution (PDI < 0.2), and the 1 wt% PEGylated DMPC/CHO 2:1 NPs were slightly smaller than those using 1 wt% PEGylated DMPC/CHO 4:1.By increasing the PEG content to 5 wt%, which has been widely used in commercial products such as in Moderna mRNA-LNP vaccine and Onpattro LNP formulation, [27] all the NPs became smaller in size compared to those NPs with 1 wt% PEG, and the DMPC/CHO 2:1 formulation displayed a smaller size than the 4:1 formulation.The surface charge of pure LNPs, 1 wt% PEGylated and 5 wt% PEGylated 40%DL DMPC/CHO 2:1 DTX-loading LNPs was −23 ± 1 mV, −18 ± 1 mV and −13 ± 1 mV, respectively.The detailed characterizations of DTX-relevant LNPs are summarized in Table S2.
Following the preliminary screening, it became apparent that all formulations containing DMPC and cholesterol with a mass ratio of 2:1 and 4:1 were suitable for producing small NPs with a narrow size distribution.When other factors were kept constant, the LNPs formulated with a DMPC/CHO ratio of 4:1 exhibited a larger size compared to those formulated with a 2:1 ratio (Table S2).Specifically, the EE% of 5 wt% PEGylated DTX-loading NPs with a DMPC/CHO ratio of 2:1 was as high as approximately 99%, and the final DL% of this formulation was 40% ± 2%.
The TEM images of the 40%DL 1 wt% PEGylated DTXloaded LNPs with the mass ratio of DMPC to cholesterol at 2:1 and 4:1 reveal that the LNPs with high DTX loading are spherical with a hydrodynamic particle size of 70-80 nm (Figure 3B).This small size (less than 100 nm) is advantageous for achieving longer circulation time and higher drug efficiency as previously reported. [3]igure 4 shows the stability of 40%DL DTX-loaded LNPs formulated with different PEG content (1 wt% and 5 wt%) and different DMPC/CHO mass ratios (2:1 and 4:1) using water, PBS and HEPES as the antisolvents at room temperature over 144 h.All LNPs using DMPC/CHO at a 2:1 mass ratio remained stable at round 100 nm during the 144-h experiment period at room temperature.The DMPC/CHO 4:1 NPs remained stable but with a bigger size (around 150 nm), and the DMPC/CHO 4:1 NPs with 5 wt% PEG prepared using water displayed a significant size increase from 78.9 ± 0.7 nm to 132.2 ± 0.3 nm after 48 h.The results indicate that PEG content did not significantly impact on NPs stability.Addi-tionally, DMPC/CHO 2:1 NPs were more uniform and evenly distributed than the DMPC/CHO 4:1 NPs (Table S2).
The TEM images in Figure 4 reveal the structure characteristics of DMPC/CHO 2:1 NPs, which appeared to be affected by the PEG content.At low PEG concentration (1 wt% PEG), the DMPC/CHO 2:1 NPs in PBS were observed to be solid and spherical particles without any visible outer layer under TEM, a morphology that was also observed in NPs produced in water as shown in Figure 3B.However, when increasing the PEG content, the DMPC/CHO 2:1 NPs exhibited a solid core that was tightly encapsulated by a lipid shell.Notably, 5 wt% PEGylated NPs had slightly smaller sizes compared to NPs with 1 wt% PEG.
Overall, the DMPC/CHO lipid mass ratio at 2:1 resulted in better DTX-loaded LNPs than that of the 4:1 ratio, in terms of both particle size and stability.Moreover, 5 wt% PEG NPs were found to be more uniform and evenly distributed than the 1 wt% PEG NPs.In contrast, the choice of the aqueous phase (water, PBS, HEPES) did not have a significant effect on the stability of NPs but did have a slight impact on the NP size.

Synthesis and characterization of LNPs with high CCM loading
To demonstrate the versatility of this new concentrationcontrolled sequential nanoprecipitation method, curcumin was used as another model drug.Similarly, control groups include free CCM, free DMPC NPs, and pure cholesterol NPs (Figure 5A).According to Table S3 free CCM alone was able to form NPs of good size around 100 nm and a narrow size distribution (PDI = 0.06) when measured within 5 min after production.However, after 5 min, free CCM NPs started to precipitate rapidly (Figure 5B). Figure 5C further demonstrates that free CCM formed NPs initially then started to precipitate out, indicating free CCM was unstable as NPs.Pure DMPC NPs had a smaller size (46.5 ± 0.2 nm), while pure cholesterol NPs had a larger PDI value of 0.4 (Table S3).
Among the formulations in Table S3, two CCM-loaded NPs using DMPC, that is, CCM-LNPs (S) and PEG-CCM-LNPs (S), were identified as the best formulations with average sizes of less than 150 nm, and the single-lipid LNP formulation exhibited a relatively smaller PDI (0.15) (Figure 5A; Table S3).The same formulation was suc-cessfully synthesized in PBS buffer as well.Instead of employing double-lipid components (D), the single phospholipid CCM-LNPs did not only have a smaller particle size (108 ± 2 nm) but also a good size distribution (PDI = 0.15).The zeta potential of this single phospholipid CCM-LNPs was −32.2 ± 0.4 mV.In contrast, the cholesterol-CCM NPs had a narrow size distribution (PDI = 0.09) but relatively larger size (171 ± 4 nm) (Table S3).Considering that curcumin is photodegradable in aqueous solutions, the stability test of CCM-loaded NPs was conducted at 4 • C with aluminium foil shielding.The stability test for each formulation was halted when visible precipitates appeared.Figure 5B shows the size change of the CCM-loaded NPs over 72 h.The size of the free DMPC NPs and cholesterol-CCM NPs increased rapidly, indicating their instability (Figure S2).Free CCM NPs showed a slow increase in size over the first 24 h, after which visible precipitation appeared at 48 h (Figure 5C).PEGylated CCM-loading NPs with double lipid encapsulated (PEG-CCM-LNPs (D)) remained stable at 200 nm over the first 48 h but started to get larger after that.Interestingly, except CCM-loaded LNPs encapsulated by DMPC only (PEG-CCM-LNPs (S)), the free cholesterol NPs, CCM-loaded LNPs encapsulated by double lipid DMPC/CHO with a mass ratio of 2:1 (CCM-LNPs (D)) remained stable, as indicated by the constant size and PDI (Figure 5C).
Figure 5C illustrates the stability of 50% CCM-loaded LNP suspensions over a period of 72 h, showing that all samples, except for CCM-LNPs (S) and PEG-CCM-LNPs (S) samples, underwent precipitation.Visible aggregation was observed for free CCM NPs and free DMPC NPs, with CCM NPs forming yellow aggregates settling at the bottom of the cuvette whereas DMPC floating at the top of the suspension.The two LNPs with 50% CCM loading, that is, CCM-LNPs (S) and PEG-CCM-LNPs (S), remained highly transparent over 72 h.On the other hand, cholesterol-CCM NPs exhibited a significant amount of precipitates and formed two distinct layers.The CCM-LNPs (D) and PEG-CCM-LNPs (D) samples appeared opaque but with an uniformally distributed yellow turbid appearance (Figure 5C).The pure cholesterol NPs sample had a gel-like and evenly dispersed milky white precipitate.Moreover, the size change of the samples (Figure 5B) further supported that the CCM-LNPs (S) and PEG-CCM-LNPs (S) were the optimal two formulations with high CCM-loading LNPs.Despite the high PDI value of PEGylated formulations in comparison with the non-PEGylated NPs, the CCM-LNPs (S) was chosen as the best formulation for further morphological analysis.Figure 5D shows the morphology of the CCM-LNPs (S) sample with 50%DL, showing spherical solid NPs.

Synthesis and characterization of CPT-loaded LNPs
CPT is a cancer treatment reagent that is well-known as a DNA topoisomerase I inhibitor.However, the poor solubility has limited its clinical applications so far. [28]CPT has very low solubility in both DMSO and EtOH solvents (Table S4), which makes it challenging to work with.Although CPT can be dissolved in DMSO at a concentration of 5 mg mL −1 after being rolling mixed overnight, aggregates formed quickly when an aqueous phase is added due to the high saturation.Therefore, a 2 mg mL −1 CPT solution in DMF or DMSO was selected as the drug stock solution.The organic solvent system was composed of DMSO, DMF and EtOH.Table S5 shows that free CPT tends to aggregate easily.In contrast, the CPT-loaded NP with a DMPC/CHO mass ratio of 2:1 showed an ideal size around 100 nm and PDI of around 0.1.However, CPT-loaded formulations have relatively low DL% (the highest DL% achieved so far was 20% DL) compared to other drug-loaded LNPs due to its poor solubility in most of the organic solvents that were attempted.
Figure 6A illustrates that the free CPT can easily get aggregated, whereas CPT-loaded LNPs using the DMPC/CHO 2:1 showed good particle size (103 ± 2 nm) and PDI (0.1 ± 0.03).Moreover, the CPT-loaded LNPs produced by the concentration-controlled sequential nanoprecipitation method remained stable during the experiment period of 72 h (Figure 6B).The 20%DL CPT-loaded LNPs without PEG exhibited solid and spherical morphology (Figure 6C).While they look slightly different in the TEM image with less particles shown after 3-month storage at room temperature, these CPT-loaded LNPs based on the DLS analysis maintained a small particle size of approximately 110 nm and PDI value (0.13) (Figure 6D).Although the NPs exhibited some drug precipitation and slight increase in particle size due to their swelling in an aqueous environment, there was no significant change in appearance in comparison with the freshly-produced NPs.If a suitable organic system with high CPT solubility is identified in the future, CPT-loaded for-mulation using DMPC/CHO 2:1 has the potential to produce LNPs with high CPT loading.

Synthesis and characterization of ibuprofen-loaded LNPs
A common but less hydrophobic drug, ibuprofen, was employed to investigate the impact of drug hydrophobicity on the encapsulation process of the sequential nanoprecipitation.As ibuprofen is fully soluble in EtOH (5 mg mL −1 ), both single-solvent or double-solvent systems could be used to synthesize ibuprofen-loaded LNPs. Figure 7 shows the properties of LNPs with 40% ibuprofen loading using different sovlent systems.The 5 wt% PEGylated ibuprofen-loaded LNPs produced with a single-solvent system had a particle size of 127 ± 1 nm (PEG-ibuprofen-LNPs (SS)), while those produced with a double-sovlent system was around 99 ± 2 nm (PEG-ibuprofen-LNPs (DS)) (Figure 7A).Both formulations have much smaller sizes than that of the pure ibuprofen NPs (203 ± 1 nm), and they have a uniform distribution in the suspension (PDI < 0.15) (Table S6).
The stability test (Figure 7B) shows that the size of free ibuprofen NPs was reduced by half, possibly due to its high water solubility.However, both the ibuprofen-loaded LNPs fabricated using single-solvent and double-solvent systems remained stable over 72 h at room temperature.In particular, the LNPs produced using a double-solvent system showed better properties in terms of size (110 nm) and size distribution (around 0.1). Figure 7C demonstrates the TEM image of freshly-fabricated spherical ibuprofen-loaded LNPs.After 3 months of storage in an aqueous phase at room temperature, the NPs maintained their spherical shape but increased in size (Figure 7D).In summary, the PEGylated DMPC/CHO 2:1 formulation is optimal for encapsulating less hydrophobic ibuprofen.

Synthesis and characterization of DOX-loaded LNPs
Another current front-line standard cancer treatment, DOX (free base), was chosen as the drug cargo.The solubility of DOX in common aqueous solutions like water and PBS was investigated firstly to ensure DOX is able to precipitate out when an antisolvent is added.Figure S3 illustrates that after being thoroughly mixed by a roller mixer overnight, all DOX powder dissolved completely in water but remained mostly insoluble in PBS buffer.The solubility of DOX in PBS at room temperature was measured to be approximately 0.2 ∼ 0.3 mg mL −1 , which was sufficient for further experiments.
Interestingly, the development of high DOX-loaded LNPs followed a similar path to that of CCM.Through a series of screening using different DMPC/CHO mass ratios (1:2, 2:1, 4:1, 1:0), it was confirmed that PEGylated single-lipid (DMPC only) 40% DOX-loaded formulations exhibited better properties (Table S7).As shown in Figure 8A, DOX alone was not able to form NPs. With the incoporation of lipid, the size of the NPs significantly reduced to one tenth of the free DOX NPs.As the proportion of DMPC in the formulation of DMPC/CHO increased, the size of DOX-loaded LNPs increased accordingly (Table S7).Nonetheless, when cholesterol was excluded from formulation, the size of NPs returned to around 100 nm with good dispersity, which is favorable for drug delivery.Furthermore, PEGylated DOXloaded LNPs remained stable up to 72 h and the EE% of this formulation was as high as 97 ± 2% (Table S8).The TEM image reveals that the sample contained two main types of NPs: core-shell structured LNPs and hollow LNPs.The white dots on the background with a size of 10 nm were resulted from UA staining (Figure S4).This concentration-controlled sequential nanoprecipitation technology provides a promising and efficient method for synthesizing LNPs with high DOX loading.

3.6
Nanoparticles with tunable drug loading using the concentration-controlled sequential nanoprecipitation method Most of the current lipid-based nanomedicines in clinical applications are fabricated by the traditional thin-film method or high-pressure homogenization method, leading to relatively low drug loading and high production cost.This easy and facile concentration-controlled method is not only suitable for synthesizing NPs with high drug loading, but also adaptable to produce NPs with different drug loading by adjusting drug-to-lipid ratio to achieve sequential nanoprecipitation.Herein, CCM and DTX were selected to examine if this method is able to synthesize LNPs with tunable drug loading.
Figure 9 shows the morphology of LNPs with varying drug loading.Plain LNPs in the presence of cholesterol formed liposomes, with a size of around 80 nm, while NPs in the absence of cholesterol were irregular due to the self-assembly of redundant lipids into aggregates at high lipid concentration.As drug/lipid mass ratio increased to 1/99 (1%DL), both CCM-loaded LNPs and DTX-loaded LNPs formed drug-loaded liposomes with drug encapsulated within the phospholipid bilayer.When the DL% increased to 20%DL, CCM-LNPs demonstrated a multi-layered soft cocoon structure and DTX-LNPs remained liposome-like but the bilayer started to get thicker and uneven due to the high drug/lipid ratio, resulting in more DTX precipitation.Furthermore, as the DL% increased further, CCM-LNPs showed no obvious change but the morphology of high DL% DTX-LNPs changed significantly to drug-core lipidshell NPs instead of liposomes.In order to further prove the drug-core lipid-shell structure of LNPs when drug loading was high, Zinc Phthalocyanaine, an organometallic dye, which is poorly soluble in water was employed as a model to observe the morphology of LNPs using Scanning Transition Electron transmission Microscopy with Eneregy-dispersive X-ray spectroscopy (STEM-EDX) tomography (Figure S5).The results confirmed the hypothesis that with a high lipid concentration, the co-precipitation of lipid and drug results in the formation of liposomes.When the drug/lipid ratio mass becomes very high, the LNPs formed a drug-core lipid-shell structure (Figure 1).Although LNPs with even higher DL% (60% or 70%) could also be produced (Table S3; Table S7), only optimal formulations with good stability were considered.Therefore, the concentration-controlled sequential nanoprecipitation technology represents a promising approach for fabricating lipid-based nanomedicines with tunable drug loading and good stability.This method enables the production of LNPs with tunable drug loading ranging from low to exceptionally high (70 wt%), and high encapsulation efficiency (EE% all above 85%).Moreover, by adjusting the drug/lipid mass ratio, we were able to generate two types of NPs with distinct drug localization.

In vitro drug release study of LNPs with high DTX loading
To further investigate the differences between high drug loading and low drug loading LNPs in terms of their in vitro release kinetics, DTX was selected as a model drug to conduct studies under sink condition.The release profiles of high DTX-loaded LNPs and low DTX-loaded LNPs were investigated in PBS buffer containing 0.2% w/v Tween 80 under 37 • C with a gentle shaking speed of 150 rpm to mimic the physiological environment.During the initial 2 h, both low DTX-loaded LNPs and high DTX-loaded LNPs exhibited similar release behaviors and achieved around 20 to 30% cumulative release.However, in the next 4 h, the low DTX-loaded LNPs exhibited a much more rapid release, with nearly 100% drug release at 6 h.In contrast, high DTX-loaded LNPs demonstrated a much slower and more sustained release, with only around 50% of DTX being released at 12 h (Figure 10), mainly due to the drug-core structure of the high DTX-loaded LNPs.As we previously demonstrated using J-aggregate-based FRET method, high drug loading NPs with a drug core structure experience two release processes, that is, the dissolution of drug core into drug molecules, then the release of drug molecules. [29]herefore, it takes extra time for the dissolution of drug core into molecules for the high DTX-loaded LNPs, thus more sustained release.

CONCLUSIONS
The present study reports the development of a new concentration-controlled sequential nanoprecipitation method for the synthesis of LNPs with high drug loading.Our findings revealed that in addition to the impacts of solvent composition, lipid concentration plays a crucial role in controlling the precipitation sequence of drug and lipid.By controlling the lipid concentration, we successfully fabricated a library of LNPs with tunable DL% ranging from 1 wt% to exceptionally high (up to 70 wt%) for a variety of drugs, including two front-line anti-cancer treatments, DTX and DOX, as well as hydrophilic molecules like ibuprofen.Moreover, we observed that the concentration of lipids affects the morphology of the LNPs, with high lipid concentration leading to the formation of low DL% liposomes and low lipid concentration resulting in a drug-core, lipid-shell high DL% LNPs.Overall, this study demonstrated a facile and highly adaptable concentration-controlled sequential nanoprecipitation method for synthesizing LNPs with tunable drug loading.To the best of our knowledge, this is the first study to fabricate LNPs with high drug loading using such a facile method.Our study provides a new understanding of sequential nanoprecipitation technology and a more controlled way to produce NPs with desirable drug loading.As demonstrated previously that high drug loading NPs exhibited enhanced cellular uptake and cytotoxicity in cancer cells, as well as improved anti-tumor efficacy with fewer side effects in mice models compared to low drug loading NPs, [7,20] future in vitro and in vivo studies will be conducted for these LNPs systems with high drug loading.Co-loading of two or multiple drugs would be beneficial for addressing some of the challenges facing combination therapy and other biomedical applications, [30,31] so future studies will be conducted for making LNPs with co-loading of dual or multiple drugs.Furthermore, stable LNP formulations need to be developed in future for their long-term storage, for example, through lyophilization.LNPs with a high drug loading have the advantage of delivering a maximum amount of drug with a minimum number of NPs, but it is critical to limit the potential off-target risk.The advance in targeted delivery would provide strategies for modifying these high drug-loading LNPs for targeting delivery.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

F
I G U R E 1 A nanoprecipitation method for forming lipid nanoparticles (LNPs) encapsulating different drugs.(A) An aqueous anti-solvent is added to an organic phase containing hydrophobic drugs and lipid components with different drug/lipid ratio to induce sequential nanoprecipitation.(B) Drugs that have been successfully encapsulated by LNPs with high drug loading include docetaxel, curcumin, doxorubicin, camptothecin and ibuprofen.(C) The lipid materials that were employed to form drug-loaded LNPs, include DMPC, cholesterol and DSPE-PEG 2 kDa.2.2.7 Data analysis All the experiments were performed in triplicate and repeated three times.Statistics analysis was computed using Graph-Pad Prism version 9.0.0 for Windows, GraphPad Software, San Diego, California USA.Standard unpaired two-tailed Student's t test was used to test for statistical significance between groups, with P < 0.001 denoted as ***p < 0.01 denoted as **p < 0.05 denoted as *.Values for p are included in the appropriate figure legend or the main text.

F I G U R E 2
Sequential nanoprecipitation of drug and lipids at different lipid concentrations.The nanoprecipitation process of drug (Docetaxel [DTX]) and lipid (DMPC) in low lipid concentration (1.76 mg mL −1 ), medium lipid concentration (2.43 mg mL −1 ) and high lipid concentration (3.10 mg mL −1 ) respectively from left to the right.(I) Drug precipitation curves with lipid concentration increasing.(II) Lipid precipitation curves with lipid concentration increasing.(III) Merged drug and lipid precipitation curves with different DL%.(A) Precipitation sequence of drug and lipid components of 40%DL lipid nanoparticles (LNPs).(B) Precipitation sequence of drug and lipid components of 20%DL LNPs.(C) Precipitation sequence of drug and lipid components of 1%DL LNPs.For the precipitation curve, Docetaxel was selected as model drug.

F I G U R E 3
Characterization of high docetaxel (DTX)-loading lipid nanoparticles (LNPs).(A) The size and size distribution of different DTX relevant formulations.All the formulations were produced in water.From left to right: pure DTX NPs; pure LNPs using DMPC (S); pure LNPs with mass ratio of DMPC to cholesterol 2:1 (D); single-lipid 40%DL DTX-loading 1 wt% PEGylated LNPs using DMPC (S); double-lipid 40%DL 1 wt% PEGylated DTXloading LNPs with the mass ratio of DMPC to cholesterol 2:1 (D); double-lipid 40%DL 1 wt% PEGylated DTX-loading LNPs with the mass ratio of DMPC to cholesterol 4:1 (D).The mean ± s.d.from three independent replicates is shown.***p < 0.001, analyzed by two-tailed Student's t-test.(b) Left: The TEM images of 1 wt% PEGylated DTX-loading LNPs with 40%DL with lipids mass ratio of 2:1.Right: The TEM image of 1 wt% PEGylated DTX-loading LNPs with 40%DL with lipids mass ratio of 4:1.The samples were dialyzed in water at 4 • C overnight before TEM analysis.Scale bar = 100 nm.

F I G U R E 6
Characterization of lipid nanoparticles (LNPs) with high CPT loading.(A) The size and size distribution of 20% CPT-loaded LNPs produced using the concentration-induced sequential nanoprecipitation method.The mean ± s.d.from three independent replicates is shown.***p < 0.001, analyzed by two-tailed Student's t-test.(B) The stability of CPT-loaded LNPs at room temperature with aluminium foil shielded for 72 h.(C) The morphology of freshly-produced 20% CPT-loaded LNPs under TEM.Scale bar = 100 nm.(D) The morphology of 20% CPT-loading LNPs after 3 months under TEM.Scale bar = 100 nm.

F I G U R E 1 0
In vitro drug release profiles of high docetaxel (DTX)-loaded lipid nanoparticles (LNPs) and low DTX-loaded LNPs.The release study was conducted under sink condition in PBS containing 0.2% w/v Tween 80 under 37 • C with shaking speed of 150 rpm.The drug loading of low DTX-loaded LNPs: 1% DL; The drug loading of high DTX-loaded LNPs: 40% DL.This study was performed in triplicate, the results were presented in mean ± s.d.
The project was supported by the Australian Research Council Projects (grant number: DP200101238) and Australian National Health and Medical Research Council projects of Australia (grant number: APP2008698).Yun Liu acknowledges the financial support from the Australian Research Council Discovery Early Career Researcher Award (DE230101044).Letao Xu would like to thank the support from Australian Government Research Training Program Scholarships.The authors acknowledge the facilities, and the scientific and technical assistance, of the Adelaide Microscopy Centre, The University of Adelaide, and the Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland.This work was performed in part at the Queensland node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano-and microfabrication facilities for Australia's researchers. ).