Cryptococcus neoformans is a life-threatening invasive fungal disease. It is more than merely a multi-organ infection; the fungus’ intracellular survival and extracellular proliferation play essential roles in the pathophysiology of C. neoformans infections. Because significant drug accumulation at target organs and cells remains a challenge, an effective delivery method is critical for treating these illnesses.
A bioresponsive micro-to-nano (MTN) device that efficiently clears C. neoformans in vivo is described here. This strategy is based on our in-depth study of the overexpression of matrix metalloproteinase 3 (MMP-3) in infectious microenvironments (IMEs) and secreted protein acidic and rich in cysteine (SPARC) in several associated target cells. In this MTN system, bovine serum albumin was employed to make nanoparticles (NPs), which were then conjugated with an unique linker that largely consisted of a BSA-binding peptide and an MMP-3-responsive peptide to make microspheres.
After intravenous administration, the MTN system was mechanically caught by the tiniest capillaries in the lungs, and MMP-3 dissolved it into BSA NPs in the IMEs. Based on the overexpression of SPARC, the NPs also targeted lung tissue, brain, and infected macrophages, reaching various targets and delivering effective therapy (Fig. 1).
Materials and Methods
Cell culture and animals
Cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 80 U/mL penicillin and 0.08 mg/mL streptomycin. All cells were kept at 37 °C in a humidified incubator with 5% CO2. C. neoformans var. grubii (serotype A) strain H99 was acquired from American Type Culture Collection and grown in nutrient-rich yeast extract-peptone-dextrose (YPD) medium at 30 °C.
Surface plasmon resonance (SPR) measurements
The interaction between the peptide and BSA was quantified by SPR (Nicoya Lifescience, Waterloo, Canada) at 25 °C. To immobilize BSA on the chip, 100 µL of 0.02 mol/L NHS dissolved in activation buffer was injected to activate the carboxyl group.
A pulse of 50 mmol/L cholamine was injected to quench redundant activated carboxyl groups. To validate the affinity between ligand and BSA, the peptide of different concentrations was injected at a flow rate of 20 µL/min with PBS. All concentrations were performed in triplicate and after each test the chips were regenerated by injection of 200 µL of 10 mmol/L HCl.
Preparation and characterization of BSA NP/AmB
NP was prepared by emulsion-solvent evaporation. Briefly, 10 mg BSA was dissolved in 1 mL 0.9% NaCl, 0.5 mg AmB was dissolved in 25 µL dimethyl sulfoxide (DMSO) and 175 µL of a chloroform and methyl alcohol mixed solvent (4:1, v/v). Then, 1 mL of BSA was mixed with 200 µL AmB (0.5 mg) to sonicate for 5 min using a bath sonicator at a power of 400 W. The resulting NP was hardened during solvent evaporation for 3 h, and free AmB was removed by gel filtration on a Sephadex G-50 column.
In vitro interaction of NP with RAW264.7 cells
Images were obtained using an Operetta CLS high-content screen imaging system C. neoformans were collected, washed twice with PBS, and opsonized with 20% mouse complement for 1 h at 37 °C and 5% CO2.
Isolation of pulmonary microvascular endothelial cells (PMVECs)
The explant method was used to isolate and culture mouse PMVECs23. After anesthesia with 10% phenobarbital, the lungs of the mice were collected. Then, the peripheral lung tissue was cut into 1 mm3 pieces, rinsed with serum-free medium three times, and evenly placed into 25 cm2 plastic culture bottles. After incubation for 2 h, 2 mL of DMEM containing 20% fetal bovine serum, 90 U/mL heparin sodium, and 50 µg/mL endothelial cell growth supplement was added for static culture. Cell morphology was observed under a light microscope.
Cell protein level detection by immunofluorescence
Cells were infected as described above and fixed with 4% paraformaldehyde for 30 min, blocked with 5% BSA and incubated overnight at 4 °C with anti-SPARC antibody, then subsequently stained with Alexa Fluor 488-conjugated secondary antibody for 1 h at room temperature, and followed by DAPI (4’,6-diamidino-2-phenylindole) staining.
Conjugation of peptide with PEG10000-Mal
The amino acid sequences of the BSA-binding peptide and MMP-3-responsive peptide (NFF-3) were integrated into a new single peptide and selected as the linker, named the PN peptide. Briefly, the PN peptide and PEG-Mal (molar ratio 5:1) were dissolved in freshly distilled dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS), respectively. The solution was stored at −20 °C.
Preparation and characterization of BSA MTN
To generate NP bound to PN-PEG, PN-PEG was first dissolved in 0.9% NaCl. Next, 4 mL of a 0.5 mg/mL NP solution was added to 1 mL of a PN-PEG (0.5 mg/mL) with stirring, incubated for 30 min at 37 °C. Size distribution was at 25 °C, and the samples were imaged using scanning electron microscopy (SEM).
Establishment of BALB/c mice model infected with C. neoformans
Cells of C. neoformans wild-type H99 strain or the RFP-expressing C. neoformans strain used for infection were cultured in YPD medium overnight at 30 °C. The medium was removed, and the cells were washed three times with PBS and diluted to 5 × 106 colony-forming units (CFU) per mL (CFU/mL). Then BALB/c mice were inoculated with 20 µL of the C. neoformans suspension via intranasal instillation to establish the infection model.
In vivo and ex vivo imaging of BSA MTN in infected mice
In vivo and ex vivo imaging of BSA MTN in infected miceFor in vivo and ex vivo real-time tracking, BSA NP, BSA MP, and BSA MTN were labeled with Near-infrared DiR fluorescent probe. After 48-h infection, the C. neoformans-bearing mice were injected with the preparations via the tail vein. In vivo images were monitored using an FX pro in vivo imaging system at predetermined time points (1, 4, 8, 12, and 24 h). For ex vivo images, mice were sacrificed at 8 h post-administration, and the major organs were carefully collected, rinsed with cold PBS, and observed.
In vivo pharmacodynamics efficacy
- After 24-h infection, model mice were randomly divid-ed into four groups and treated intravenously with saline, BSA NP/AmB, BSA MP/AmB, and BSA MTN/AmB respectively, with a single dose.
- The survival rates for all mice were monitored for 42 days after treatment.
- After 24-h infection, infected mice were treated with saline, BSA NP/AmB, BSA MP/AmB, and BSA MTN/AmB at 2 mg/kg of AmB respectively.
- On Days 3 and 7 after treatment, mice were sacri-ficed, and then the lung and brain tissues from each group were carefully collected, weighed, and homogenized with sterile saline.
- The homogenate was continuously diluted by sterile saline, and then 50 µL of the suspension was inoculated onto YPD medium with 1% penicillin–streptomycin (1:1, v/v) and incubated at 30 °C for 48 h to determine CFU/g of lung tissue and CFU/g of brain tissue.
- On the tenth day after treatment, animals were anaesthetized with isoflurane gas and MRI examinations were carried out at a 1.0 T MRI scanner, and the mean signal intensity in the lung area was measured by Image-Pro Plus.
- Multiple group comparisons were conducted using one-way analysis of variance (ANOVA).
- All data were analyzed using IBM SPSS Statistics 20.0.
- All data are presented as the mean ± SD. Differenc-es were considered significant at *P < 0.05, **P < 0.01, and ***P < 0.001.
Results and discussion
Design and characterization of the MTN system
One of the major design considerations of the MTN system is the ability to transform between NPs and microspheres. In order to effectively assemble the NPs into microspheres, we chose 4-arm-PEG10,000-MAL The linker consists of two parts, a ligand, which is a polypeptide fragment that binds to the albumin NPs and responds quickly to MMP-3, and a multi-armed PEG. Based on these findings, we integrated the amino acid sequence of the BSA-binding and NFF-3 peptides into a new single peptide and named it PN. Multi-armed PEG is a star-like structure carrying multi-hydroxyl or functional groups, which are nontoxic, non-immunogenic, non-antigenic and amphiphilic27. (4-arm-PEG-Mal) as a structural carrier, and the successful attachment of the PN-PEG was verified by MALDI-TOF mass spectrometry (Fig. 2).
In vitro response and targeting of the MTN system
After incubation with MMP-3 (0.15 µmol/L) at 37 °C, the average size of the BSA MTN/AmB decreased from 7 µm to 115 nm within 2 h, which was further confirmed by scanning electron microscope (SEM) analysis. As a control, the BSA MP/AmB was prepared in the same way, except that the MMP-3-responsive peptide was replaced by a scrambled peptide.
These results demonstrate that the size change triggered by MMP-3 occurred efficiently, and this responsiveness was specific to the MTN system. Therefore, we first evaluated whether BSA NPs released from “shrinkable” BSA MTN could specifically bind to infected macrophages. Further, we found that this targeting preference was due to the significant upregulation of SPARC by macrophages after infection.
The uptake of BSA NPs was decreased when the infected cells were blocked with anti-SPARC antibody or silenced with SPARC siRNA.This suggests that overexpressed SPARC is beneficial for the cell-specific delivery of BSA NPs. In addition, we investigated the intracellular distribution of NPs in the macrophage-like cells. After intravenous injection, the penetration of NPs relies on the corresponding vascular endothelial cells of the lungs and brain.
We found high expression of SPARC in infected PMVECs by immunofluorescence analysis, and the uptake efficiency of the infected PMVECs was significantly higher than that of uninfected PMVECs. BSA NP showed substantial penetration in the cell culture model, which may be attributed to the high level of SPARC expression on the infected bEnd3 cells. These results indicate that BSA MTN has good responsiveness to MMP-3 in vitro (Fig. 3).
In vivo distribution and targeting of BSA MTN
To verify whether the BSA MTN can be specifically accumulated in the lungs, we used a qualitative imaging assay to investigate the in vivo and ex vivo targeting efficiency of BSA MTN/Dir. At all sampling time points, we found that the BSA MTN/AmB group showed significantly higher drug accumulation in lung tissue than the BSA NP/AmB group did, especially at 4 h post-administration.
We further explored the in vivo targeting behavior of the BSA MTN that “shrinks” to NPs via MMP-3 activity in IMEs. In addition to the passive targeting that played a major role in the mechanism responsible for preferential accumulation in the lungs, active-targeting mechanisms were also involved for albumin-based lung penetration and brain delivery. We found that both the lung and brain blood vessels highly expressed SPARC, which was demonstrated by clear colocalization of CD31 and SPARC.
More importantly, BSA NPs showed a significantly enhanced penetration in the infected lung and brain, and SPARC overlapped with the distribution of the NPs, demonstrating the importance of the SPARC-mediated uptake mechanism. By combining passive and active targeting, MMP-3-responsive MTN system can target multiple sites, which is consistent with our in vitro MTN studies (Fig. 4).
Therapeutic efficacy of BSA MTN/AmB
In addition, in vivo experiments demonstrated reduced C. neoformans infection as well as prolonged survival time with BSA MTN/AmB treatment. Four groups of infected mice (n = 10 per group) received two doses of saline solution or 2 mg/kg of preparation loaded with AmB.
Within the 42-day observation period, we found that the survival rate of the mice treated with BSA MTN/AmB was significantly higher than that of the other treatment groups. Compared with the BSA NP/AmB group, the colony-forming units (CFU) in the lungs and brain of the model animals were significantly reduced after BSA MTN/AmB treatment for 3 and 7 days. Magnetic resonance imaging (MRI) was performed to visualize the lung infection in vivo.
In the pulmonary lesions, we found that the untreated infected mice exhibited high signal intensities (white) on MRI due to inflammation, whereas the BSA MTN/AmB group exhibited no inflammatory signals. This implies a complete recovery in the latter group, which was superior to the results obtained from the BSA NP/AmB and BSA MP/AmB groups.
In view of the fact that the lung is the core invasion organ for many infectious diseases, this study is expected to provide useful insights for treating many lung diseases, especially intracellular infections. The successfully designed new DDS, BSA MTN, is responsive to MMP-3 in IMEs and combines passive and active targeting strategies against complex fungal infections. In addition, the full use of albumin plays an essential role, in which it can serve as both a drug carrier and targeting ligand, imbuing the constructed delivery system with good drug-loading capacity and efficiency.
Source: Liting Cheng, Miao-Miao Niu, Tong Yan, Zhongyi Ma, Kexin Huang, Ling Yang, Xin Zhong, Chong Li, Bioresponsive micro-to-nano albumin-based systems for targeted drug delivery against complex fungal infections,Acta Pharmaceutica Sinica B,Volume 11, Issue 10,2021,Pages 3220-3230,ISSN 2211-3835, https://doi.org/10.1016/j.apsb.2021.04.020.