Artificial Organelles with Digesting Characteristics: Imitating Simplified Lysosome‐ and Macrophage‐Like Functions by Trypsin‐Loaded Polymersomes

Abstract Defects in cellular protein/enzyme encoding or even in organelles are responsible for many diseases. For instance, dysfunctional lysosome or macrophage activity results in the unwanted accumulation of biomolecules and pathogens implicated in autoimmune, neurodegenerative, and metabolic disorders. Enzyme replacement therapy (ERT) is a medical treatment that replaces an enzyme that is deficient or absent in the body but suffers from short lifetime of the enzymes. Here, this work proposes the fabrication of two different pH‐responsive and crosslinked trypsin‐loaded polymersomes as protecting enzyme carriers mimicking artificial organelles (AOs). They allow the enzymatic degradation of biomolecules to mimic simplified lysosomal function at acidic pH and macrophage functions at physiological pH. For optimal working of digesting AOs in different environments, pH and salt composition are considered the key parameters, since they define the permeability of the membrane of the polymersomes and the access of model pathogens to the loaded trypsin. Thus, this work demonstrates environmentally controlled biomolecule digestion by trypsin‐loaded polymersomes also under simulated physiological fluids, allowing a prolonged therapeutic window due to protection of the enzyme in the AOs. This enables the application of AOs in the fields of biomimetic therapeutics, specifically in ERT for dysfunctional lysosomal diseases.


Gel Permeation Chromatography (GPC
Psomes C samples were separated by following parameters (separation method B): the separation starts with an isocratic step with a cross flow rate (Fx) of 2.5 mL min −1 for 5 min followed by an exponential Fx gradient from 2.5 to 0.15 mL min −1 within 50 min. The last step proceeds without Fx (0 mL min −1 ) for 30 min.

Conformation analysis by AF4-MALS
Scaling parameter: By plotting Rg vs M, ν can be determined by the slope of the curve. It gives information about the molecular shape in the used solvent Rg = K·M ν ν = 0.33 (sphere); ν = 0.5 -0.6 (random coil macromolecule); ν= 1 (rigid rod) [1]  Statistical analysis. One-way ANOVA statistical analysis was performed to evaluate the significance of the experimental data. A value of 0.05 was selected as the significance level, and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively.
The mixture was degassed using two freeze-pump-thaw-cycles and flushed with Argon. Then, CuBr (15 mg; 0.1 mmol) was added and the mixture was degassed using three freeze-pump-thaw-cycles again, backfilled with argon and stirred for 17 h at 50 °C. To abort the polymerization reaction, the mixture was diluted in 3 mL THF and with additional THF filtrated over activated neutral aluminum oxide to remove any copper species. From the resulting gloomy solution, the solvent was transferred to a dialysis membrane (regenerated cellulose, MWCO 5 kDa) and was dialyzed against methanol (technical grade) for three days exchanging the solvent twice a day before it was dried in vacuum to give a sticky polymer (79 % yield). The composition and the number average molecular weight (Mn) of the block copolymers were determined with 1 H NMR spectroscopy from the peak integrals. Additionally, the molar mass distributions (Ð) were determined by SEC as described in previous section. Table S1 and  by fitting of the DLS data ( Figure S10-11, Figure S24).

Control experiments using free Trypsin (Tryp) a) Trypsin Activity Colorimetric Assay Kit
The technical bulletin of the kit from Sigma-Aldrich is referred in the enzyme experiment.
Trypsin is a member of the serine protease family. It cleaves proteins and peptides into smaller pieces by hydrolyzing peptide bonds at the carboxyl side of lysine and arginine residues.
Trypsin is produced by the pancreas as an inactive trypsinogen and is then secreted into the small intestine, where it is cleaved by enteropeptidase and becomes activated. Trypsin activity aberration is implicated in gastrointestinal disorders such as pancreatitis and intestinal mucosal pathology.
Trypsin Activity Colorimetric Assay Kit offers a rapid and sensitive way to determine the trypsin activity in mammalian cell/tissue lysates, serum, plasma and other biological fluid samples. Briefly, trypsin cleaves the substrate and releases p-nitroanilide (pNA), a chromophore that can be measured at 405 nm using a spectrophotometer.

c) pH influence on the enzyme activity and stability
A Tryp stock solution (0.2 mg mL -1 ) in Milli-Q water was prepared and incubated for 1-2 h, afterwards it was filtrated using 0.2 μm filter. Then Tryp solution was diluted at different pH (6, 7 and 8) in 1 mM PBS buffer (300 ng mL -1 ) and incubated for 1 hour or 1 day. Afterwards, the Tryp activity was studied directly (Figure S13).

d) Stability study using enzyme assay and DLS
Trypsin was dissolved in 1 mM PBS buffer at pH 7.4 (1 mg mL -1 , filtrate using 0.2 μm filter) and incubated in the room temperature by slightly stirring. The hydrodynamic diameter of free trypsin was studied by DLS after 1, 2 and 3 days (Table S4). Its residual enzyme activity was studied after 1, 3 and 7 days (Figure S14). According to the calibration curves by UV-VIS spectroscopy, every trypsin has been labelled 1.6 RhB for RhB-Tryp, every trypsin has been labelled 0.8 Cy5 for Cy5-Tryp. (Figure S17). Release study using purified dye-labelled Tryp-Psomes. To check the stability and Tryp retention in purified dye-labelled Tryp-Psomes, the release behaviour was studied by dialysis (1000 kDa membrane) using purified labelled Tryp-Psomes by HFF. The sample (1 mg mL -1 ) was dialyzed in 1 mM PBS buffer at pH = 7.5 for 24 h, then 1 mL of sample was taken. Later, the sample was dialyzed in 1 mM PBS buffer at pH = 7 for 24 h, then 1 mL of sample is taken.

Fabrication and characterization of labelled Tryp-Psomes by
Finally, the sample was dialyzed in 1 mM PBS buffer at pH = 6 for 24 h, then 1 mL of sample is taken. After each process, the residual loading efficiency was studied by fluorescence intensity by triplicate ( Figure S18). The parameter for measurement: RhB: λex/λem 543/580, Gain: 80-100, Excitation Bandwidth: 9 nm, Emission Bandwidth: 20 nm.

Fabrication and characterization of Tryp-Psomes: Loading efficiency
and stability by enzyme assay Fabrication [5] . 9 mg of BCP were dissolved in 8 mL of HCl 0.01 M (pH 2, 1.12 mg mL -1 ). The solution was passed through a 0.2 µm nylon filter to remove all impurities. Next, to 7. Enzyme activity: Stability. The enzyme activity of purified Tryp-Psomes (fresh sample) was studied in comparison with three samples storage at room temperature, at 4 ℃ and at -20 ℃ for 3 days. After that, additional storages times were investigated: a) Short term storage: at 4 ℃ for 1, 3 and 5 days, B) Longer term storage: at -20 ℃ for 1, 2, 3 and 4 weeks ( Figure S20). The study was carried out at pH 7.5 by triplicate.
Enzyme activity: pH dependence. The enzyme activity of purified Tryp-Psomes (fresh sample) was studied at pH 8, 7 and 6 in 1 mM PBS buffer ( Figure S15). The enzyme activity of purified Tryp-Psomes (fresh sample) was studied at pH 7.5 and 6.5 in 1 mM PBS buffer ( Figure S16). The study was carried out by duplicate.
Enzyme activity: loading efficiency. The enzyme activity of Tryp-Psomes at pH 7.5 before and after HFF purification was studied. In order to study the loading efficiency after HFF, a calibration curve was carried out using the unpurified sample: Dilute Tryp-Psomes (0.5 mg mL -a series of concentrations sample which included 2, 4, 6, 8 and 10 μg mL -1 Tryp. Finally, the enzyme activity of the mentioned samples and HFF purified Tryp-Psomes (0.5 mg mL -1 Psomes, 0.1 mg mL -1 Tryp) were studied. Use the △A405 value of mentioned series solution to do the calibration curve. The loading efficiency of the Tryp-Psomes A and Tryp-Psomes C after HFF purification is 9.32 % and 9.02 % calculating from the calibration curve ( Figure S19). The study was carried out by duplicate.

Structural parameters of Tryp-Psomes A by AF4
The following samples were studied by AF4 (Figure S21-22

The degradation of myoglobin (Myo)/horseradish peroxidase (HRP) in the presence of free trypsin Degradation of Myo
All stock solutions were stored at 4 ℃ for not more than two weeks. The buffer was always filtrated using 0.2 µm filter before each usage.
The samples were incubated in the dark for 24 h.
Residual enzyme activity of Myo. An aliquot of 300 µL of the prepared solutions was treated with 3 µL of Amplex red (0.02 mg mL -1 in Milli-Q water) and 3 µL of H2O2 (0.02 M in Millipore). The mixture was vigorously shaken and after 15 min, the fluorescence spectra were recorded at an excitation wavelength of 534 nm was recorded immediately. Each assay was executed in triplicates ( Figure S23).

Degradation of HRP
All stock solutions were stored at 4 ℃ for not more than two weeks. The buffer was always filtrated using 0.2 µm filter before each usage.
The samples were incubated in the dark for 24 h.
Residual enzyme activity of HRP. An aliquot of 300 µL of the prepared solutions was treated with 3 µL of H2O2 (0.02 M in Millipore) and 3 µL of ABTS (0.02 M in Millipore). The mixture was vigorously shaken and after 30 min the UV absorbance at 405 nm was recorded immediately, each assay is executed in triplicates ( Figure S23).
to adjust all the samples pH to 7.5. After preparation, the UV monitoring at 405 nm was started and data points were recorded immediately, each assay is executed in triplicates (Figure 3).

Preparation of simulated blood fluids
1.13.1 Preparation of simulated blood plasma [7] 50 mL of simulated blood plasma were prepared, at pH = 7.3 (adjust it if needed  (Reference from: https://www.alzet.com/guide-to-use/preparation-of-artificial-csf/). Remark: In this paper, we name artificial cerebrospinal fluid as simulated cerebrospinal fluid buffer (SCF buffer). The incubation and measurement were the same as the mentioned above (Table 2). Figure S1. 1

Calculation explanation for BCP-A composition:
The intensity of signal "b" from the PEG part is taken as a reference, because of the known total amount of ethylene glycol units (45 units). Since PEG has a symmetric structure, the integral of peak "b" represents 4 H atoms per monomer repeating unit. In contrast, the integrals of the signals "a" referring to the whole hydrophobic block of BCP, represent 2 H atoms each; the integrals of the signals "c", referring to DMIBMA, represent 2 H atoms each; the integrals of the signals "e+f", referring to DEAEMA, represent 6 H atoms each; the integrals of the signals "d", referring to -CH3, represent 3 H atoms. The integrals of "a", "b", "c", "d" and "e+f" were

Calculation explanation for BCP-C composition:
The intensity of signal "b" from the PEG part is taken as a reference, because of the known total amount of ethylene glycol units (45 units). Since PEG has a symmetric structure, the integral of peak "b" represents 4 H atoms per monomer repeating unit. In contrast, the integrals of the signals "a" referring to the whole hydrophobic block of BCP, represent 2 H atoms each; the 21 integrals of the signals "c", referring to DMIBMA, represent 2 H atoms each; the integrals of the signals "e+f+g", referring to DMAEMA and DEAEMA, represent 8 H atoms each; the integrals of the signals "h", referring to DMAEMA, represent 6 H atoms each; the integrals of the signals "d", referring to -CH3, represent 3 H atoms. The integrals of "a", "b", "c", "d",              HFF purification can be calculated as 9.319 μg mL -1 ÷ 0.1 mg mL -1 × 100% = 9.32% and 9.015 μg mL -1 ÷ 0.1 mg mL -1 × 100% = 9.02%.         Remark: Not only we did replace the UV lamp with the new one during this study, we also replaced the conduit for light channel with the new one. The damaged conduit also affects the efficiency of photocrosslinking. Table S3. The hydrodynamic diameter of Empty-Psomes C (1 mg BCP-C mL -1 ) and Tryp-Psomes C (1 mg BCP-C mL -1 and 0.2 mg Tryp mL -1 ) before and after crosslinking (CL).