Preparation of FCS/protein nanocomplexes and ex vivo evaluation of their transdermal abilities
FCS was synthesized following a previous report22. Briefly, perfluoroalkyl carboxylic acid (PFCA) was grafted to cationic polysaccharide CS through amide coupling at a fluorocarbon substitution of ~4.9% (Figure S1). Then, FCS was mixed proteins such as immunoglobulin G (IgG) and ovalbumin (OVA) at different mass ratios for 30 minutes under mild shaking to form nanocomplexes (Figure 1a). As shown in Figure 1b & d, both FCS/IgG and FCS/OVA with mass ratio at 1:1 showed sizes around 100 nm in the transmission electron microscopy (TEM) images, consistent with their hydrodynamic diameters measured by dynamic laser light scattering (DLS) (Figure 1c & e). The zeta potentials (ZP) of both FCS/IgG and FCS/OVA showed high positive charges, which increased from 6.97mV to 30.53mV and 4.38 mV to 36.73 mV, respectively, as the increase of FCS contents during the formation of nanocomplexes (Figure 1e). Then, the circular dichroism (CD) spectra were used to verify the structure of proteins before and after forming nanocomplexes. As shown in Figure 1f, FCS/IgG showed similar CD spectrum to that of free IgG, indicating that the structure of protein remained nearly unchanged during after formation of such FCS-containing nanocomplexes. Similar result also was found in the comparison between FCS/OVA and free OVA. Additionally, the antibody affinity of aPDL1 in the formulation of FCS/aPDL1 remained nearly unchanged as measured by the competition-enzyme-linked immunosorbent assay (ELISA) (Figure S2), further demonstrating that the formation of nanocomplexes wound not affect the activity of contained proteins.
Then, the transdermal kinetics and related mechanisms were investigated. Firstly, the standard Franz diffusion system was used to measure the transdermal delivery efficiency of FCS-containing nanocomplexes across the mouse skin layer. Briefly, fresh skin tissues were fixed between two glass cells, and then FCS/IgG or FCS/OVA, in which IgG and OVA were labelled with fluorescein (FITC), were added into the donor chamber. The transmitted IgG or OVA was measured by collecting liquid samples in the receptor chamber at different time points to measure the FITC fluorescence (Figure 1g). The transdermal delivery efficiencies of FCS/protein nanocomplexes with different feeding ratios (m/m) were measured. As shown in Figure 1h, FCS/IgG with the mass ratio at 1:1 showed the highest penetration ability across the skin, which may be contributed to the fact that FCS/IgG prepared at 1:1 showed the smallest sizes compared with those prepared at other mass ratio groups. It could be clearly observed that the zeta potentials of FCS/IgG nanocomplexes increased along with increasing the ratio of FCS, which may be beneficial for skin penetration. However, further increase of FCS might lead to the aggregation of nanocomplexes. On the other hand, when the amount of IgG increased, the zeta potential of FCS/IgG nanocomplex turned to be negative with aggregation, which is also unsuitable for skin penetration. Similarly, FCS/OVA with the mass ratio at 1:1 showed the highest penetration ability (Figure 1i). Therefore, all the data above confirmed the successfully transdermal delivery of FCS/protein nanocomplexes.
Transdermal mechanism of FCS-containing nanocomplexes
Next, we investigated the underlying transdermal mechanism of such FCS-containing nanocomplexes. According to previous literature, there are three classical permeation routes for transdermal delivery including intercellular, transappendageal and transcellular routes23. Firstly, we studied the intercellular route, by which the nanocomplexes could enlarge the space between epidermis cells and thus pass through them24. During the enlargement of intercellular space, we would expect the changes of cell resistance, as well as the expression and allocation of related proteins 25,26. In this case, human skin dermis cells HACAT were used to form a cell monolayer and the transepithelial electrical resistance (TEER) between the two sides of monolayer was monitored (Figure 2a). As shown in Figure 2b, the cell single layer was formed 6 days later with stable and high TEER. Interestingly, an obvious decrease of TEER was observed with the addition of FCS/IgG on day 11, indicating the destruction of the cell monolayer and opening of intercellular channels after adding FCS-containing nanocomplexes. More interestingly, the re-increase of TEER was observed 4 hours later after the removal of FCS/IgG, and returned to its original level in 12 hours, demonstrating that FCS/IgG only temporarily opened the intercellular channel. TEM imaging of the skin also revealed the opening of the tight junctions and enlarged intercellular spaces after treatment with FCS/IgG compared with the normal skin (Figure 2 & S4). To confirm the enlargement of interstellar spaces, the changes of tight junctions (TJs) related protein such as zonula occludin (ZO)-1 were further evaluated27. With the addition of FCS/IgG, while the total expression of ZO-1 remained nearly unchanged, their continuous distribution was distinctly disturbed, indicating the opening of tight junctions along the interface between cells (Figure 2c & 2d). Moreover, the phosphorylation of myosin light chain, an important parameter for cytoskeletal structure28, was found to be up-regulated in FCS/IgG treated cells, demonstrating that FCS was able to promote the phosphorylation of myosin light chain to induce the contraction of actin and the rearrangement of cytoskeleton (Figure 2e).
In addition to the enhanced intercellular bypass permeability of FCS-containing nanocomplexes, the transappendageal pathway, which usually plays an important role in the transport of large and water-soluble drugs through the hair follicles, sweat glands and sebaceous glands, was also investigated in our experiments 16. With the counter-staining of Keratin (Krt) 14, the hair follicles and sweat glands in deep dermis region were labeled. As shown in Figure 2g (white arrows), it was observed that FCS/IgG colocalized with hair follicles and sweat glands, indicating that the tranappendageal pathway also played an important role in FCS-based transdermal delivery systems.
We further studied the transcellular route, which signifies the passage of drugs directly across keratinocytes29. Different from normal tissue cells which decompose drugs in lysosomes, polarized cells such as keratinocytes sometimes might expel drugs through exocytosis24,30. Apical exocytosis was used to investigate this phenomenon in vitro. Briefly, HACAT cells were incubated with FCS/IgG-FITC for endocytosis. After 12 h incubation, FCS/IgG-FITC were washed away and the cells were incubated for another 12 h. Then, the fluorescence of FITC in the supernatant was measured to evaluate the apical exocytosis. As shown in Figure 2h, the apical exocytosis rate of FCS/IgG-FITC from HACAT cells was less than 2%, almost the same compared with that treated with free IgG-FITC, indicating negligible transcellular route was involved. Therefore, the transdermal delivery of FCS-containing nanocomplexes mainly relied on the intercellular and transappendageal pathways (Figure 2i).
Topical application of FCS/antibody nanocomplexes for melanoma treatment
Melanoma, one of the most common malignant tumors especially among Caucasians, poses an important threat to people's life and health31. For melanoma treatment, immunotherapy especially immune checkpoint blockade using anti-programmed death-1/its ligand (aPD1/aPDL1) antibodies has achieved excellent therapeutic efficacy in clinic32. Despite the exciting therapeutic result using aPD1/aPDL1 antibodies for the treatment of melanoma, there are still many limitations such as the risk of autoimmune diseases after intravenous injection33. Considering that FCS could act as the efficient transdermal delivery carrier for proteins, we thus used it as the transdermal delivery platform to deliver aPDL1 antibody for melanoma treatment (Figure 3a). It was expected that the transmitted aPDL1 could block the PD1/PDL1 pathway to stimulate cytotoxic T cells and lead to remarkable inhibition of tumors.
To examine the topical penetration behavior of antibodies complexed with FCS, tumors from the mice swiped with cream containing radioisotope 125I labeled IgG (125I-IgG) or FCS/125I-IgG were collected for quantification of transmitted antibodies at different time points. As shown in Figure 3b, compared with free 125I-IgG in the transdermally applied cream, FCS/125I-IgG showed dramatically higher accumulation in tumor, while the radioactivities in other organs appeared to be much lower. Meanwhile, we found that the tumor accumulation of FCS/IgG showed the peaked level at over 120 % of injected dose per gram tissue (ID/g) at 12 h post the cream was applied (Figure 3c). As the cream was removed at 12 h, the IgG level in the tumor decreased slightly at 24 h. Meanwhile, the ELISA essay was also conducted to detect the tumor accumulation of IgG in the FCS/IgG formulation topically applied onto tumors in the cream. As shown in Figure S5, the ELISA essay showed similar results with the radiolabeling-based biodistribution data that IgG could be efficiently delivered into the tumor within 12 h with the aid of transdermal delivery platform using FCS/IgG nanocomplexes. On the other hand, the tumors with FCS/IgG-Cy5.5 were imbedded for tumor slicing. As illustrated in Figure 3d, the fluorescent signals of IgG-Cy5.5 in the tumor were gradually increased and evenly disbursed inside the whole tumor within 12 h, demonstrating the continuous transmission of antibodies from the FCS/IgG-Cy5.5 cream into the tumor.
Inspired by the effective accumulation of IgG in the tumor after transdermal delivery, we then carried out in vivo treatment for melanoma tumors by transdermal delivery of FCS/aPDL1. Mice bearing melanoma tumors were randomly divided into four groups: i. Untreated, ii. Free aPDL1 by intravenous (i.v.) injection, iii. CS/PDL1 in the cream by transdermal delivery and iv. FCS/aPDL1 in the cream by transdermal delivery. For transdermal delivery, CS/aPDL1 or FCS/aPDL1 solution was mixed with blank cream, and then applied on the tumor which was subsequently covered with a 3M® transparent film. Such treatment was repeated every two days for three times at the dose of 20 μg aPDL1 per mouse each time. For i.v. injection, 20 μg aPDL1 was administrated into each mouse every two days for three times. As another control, aPDL1 was mixed with non-modified chitosan (CS/aPDL1) and added into the cream for topical application. As shown in Figure 3e, the tumors in FCS/aPDL1 treated group were successfully inhibited in a short time after the third treatment, while both CS/aPDL1 group and free aPDL1 group showed negligible anti-tumor efficacy. During 20 days of observation, the FCS/aPDL1 treated group exhibited the lowest tumor growth rate and the longest survival (Figure S6).
To understand the immune activation mechanisms of such transdermally delivered immunotherapy, tumors were collected from different groups on day 12 to investigate different types of immune cells especially T cells by flow cytometry. As shown in Figure 3f, for FCS/aPDL1 treated group, the percentages of both CD4+ and CD8+ T cells showed obvious increase in the tumor, indicating that aPDL1 was successfully delivered into the tumor to revert the T cell exhaustion. As Granzyme B is important for cell programming death triggered by CD8+T cells, Ki67 is a cell proliferation, and Interferon γ is a cytokine associated with pro-apoptotic and antitumor mechanisms34, we used these three markers to analyze the activities of CD8+ cells. As can be seen in Figure 3g – 3i, all of the three markers in CD8+ T cells were increased in the FCS/aPDL1 group, suggesting the effective infiltration and activation of cytotoxic T lymphocytes (CTLs) in those tumors. For i.v. injection of aPDL1 and transdermal delivery of CS/aPDL1 groups, the tumor infiltrations of both CD4+ and CD8+ T cells as well as the expressions of granzyme B, Ki67 and IFN-g remained nearly unchanged, which might be resulted from the low tumor accumulation of aPDL1 after intravenous injection or transdermal delivery using CS. These results clearly indicated that, compared to i.v. injection of aPDL1, transdermal delivery aPDL1 using FCS resulted in enhanced antitumor immune responses.
Our platform could also be utilized to co-deliver different therapeutic proteins. In addition to aPDL1, anti-cytotoxic T-lymphocyte-associated protein 4 (aCTLA4) is another important antibody for immune checkpoint blockade to disable regulatory T cells (Tregs) and promote effective T-cell activation35,36. Thus, we further investigated the combination therapy using aPDL1 and aCTLA4 co-delivered by the transdermal delivery carrier FCS (Figure 4a). In this experiment, B16F10 cancer cells were inoculated on the right ﬂank of each mouse as the primary tumor, and a second tumor was inoculated on the opposite site of the same mouse to mimic the cancer metastasis. Three days later, mice bearing two melanoma tumors were randomly divided into five groups: i. Untreated, ii. Free aPDL1 and aCTLA4 (i.v.), iii. FCS/aPDL1, iv. FCS/aCTLA4, v. FCS/aPDL1/aCTLA4. All the groups were administrated for three times with 20 μg aPDL1 and 20 μg aCTLA4 per mouse each time. It is worth pointing out that after the second i.v. injection of aPDL1 + aCTLA4, half of mice in this group died, likely due to the horrible side effects (e.g. cytokine storm) triggered by systemic administration of both aPDL1 and aCTLA4. In contrast, the other groups with transdermal delivery of immune checkpoint blockade antibodies showed negligible abnormality. Excitingly, compared to FCS/aPDL1 or FCS/aCTLA4 treated mice, noteworthy synergistic therapeutic effect was achieved by combination therapy with transdermally delivered FCS/aPDL1/aCTLA4 (Figure 4b & 4c). More interestingly, the growth of distant tumors in mice with FCS/aPDL1/aCTLA4 treatment also was inhibited (Figure 4d & 4e). To understand the abscopal effect induced by FCS/aPDL1/aCTLA4, the distant tumors were collected 12 days after different treatments and analyzed by flow cytometry. Consistent with the above results, the number of CD8+ T cells especially Ki67+CD8+ T cells exhibited obvious increase in the second tumor (Figure 4f & 4i), and the percentages of Tregs were decreased in FCS/aPDL1/aCTLA4 treated group (Figure 4j).
The excellent anti-tumor efficacy and the increased CTLs in the distant tumor might be attributed to the following mechanisms. Firstly, the activation of CTLs in the local tumor could induce immunogenic death of tumor cells and trigger the chronic exposure of damage-associated molecular patterns (DAMPs) as well as tumor antigens. Antigen presenting cells (APCs) are then activated and subsequently present tumor antigens to CD8+ T cells, further amplifying systemic antitumor immunity to attack distant tumors. Lastly, as reported, the blockade of PDL1 in the tumor-draining lymph nodes (TDLNs), which may be more efficient to be reached by transdermal delivery, could effectively propel systemic anti-tumor T cell immunity even in distant tumor sites3738. Therefore, our transdermal immune checkpoint antibody delivery could achieve remarkable antitumor efficacies against both in local and distant tumors.
Topical application of FCS/S1/polyIC nanocomplexes for transdermal vaccination
Vaccine is another area of great interest for transdermal delivery39. For transdermal vaccine, in addition to avoiding syringe by health professionals, it could improve immune responses by targeting abundant immune cells beneath the epidermis layer. Since the outbreak of Coronavirus Disease 2019 (COVID-19), various vaccines have been developed to suppress the infection of SARS-CoV-240–42. Up to now, more than 180 SARS-CoV-2 vaccines are under research worldwide, and 26 SARS-CoV-2 vaccines are in the stage of clinical trials43. However, all these vaccines need subcutaneous or intramuscular injection, which not only require well-trained medical staffs, but also face some additional issues such as disposal of a large number of sterile syringes. The transdermal SARS-CoV-2 vaccine, on the other hand, may offer great assistance to prevent COVID-19 by self-administration, especially in areas with tight medical resources. Considering the rapid global spread of COVID-19, and the efficient transdermal delivery of antibody using FCS, we further explored whether FCS could be utilized to transdermally deliver SARS-CoV-2 vaccine in a proof-of-concept study.
To synthesize FCS-based subunit SARS-CoV-2 vaccine, FCS were mixed with the S1 subunit of spike protein of SARS-CoV-2 virus and polyIC, forming the transdermal SARS-CoV-2 vaccine (FCS/S1/polyIC) (Figure 5a). Note that poly IC as a double-strand RNA is a toll-like receptor 3 (TLR 3) agonist that has been commonly used as immune adjuvant44. We also optimized the ratio of FCS : antigen : PolyIC in the FCS/S1/PolyIC vaccine. In this experiment, OVA was used as a modulate antigen for the optimization of formulation. As shown in Figure 5c & d, FCS/OVA/polyIC with different mass ratio showed variant sizes at about 200 nm and increased zeta potential with the increase of FCS. For the skin penetration ability measured by Franz diffusion system in Figure 5e, FCS/OVA/PolyIC prepared at the mass ratio at 2:1:1 with relatively small sizes showed the highest skin permeability, and thus this formulation was used for further study.
For the in vivo vaccination experiments, mice were randomly divided into three groups: i. untreated, ii. transdermal delivery of FCS/S1/polyIC, and iii. subcutaneous (s.c.) injection of S1/polyIC. As shown in Figure 5b, mice in transdermal delivery group were administrated 3 times in 2 weeks (the doses of S1 and polyIC were both 20 μg per time), while mice in s.c. injection group were injected twice with the S1 protein dose at 20 μg per time and the polyIC dose at 50 μg per time. Interestingly, mice with transdermal delivery of FCS/S1/polyIC showed almost similar antibody titer to that of mice with s.c. injection of S1/polyIC in two weeks, indicating that the transdermal delivery of FCS/S1/PolyIC could result in almost the same B cell activation level compared with s.c. administrated vaccines (Figure 5f). Furthermore, after boosting on day 14, the specific antibody titers in both transdermal delivery group and s.c. injection group reached up to 104 in 30 days, further indicating the effective activation of humoral immunity by those vaccines. After another boost on day 42, mice vaccinated by either transdermal delivery of FCS/S1/polyIC or s.c. injection of S1/polyIC showed remained at high levels of anti-S1 antibody titers.
In addition to the activation of humoral immunity by generating specific antibody against the S1 protein, the cell immunity also plays an important role in virus clearance by training cytotoxic T cells to recognize and kill virus-infected host cells 45–47. Therefore, the levels of cytotoxic T cells in mouse spleen were evaluated on day 28 after the primeval administration. Although no obvious change of CD4+ and CD8+ T cell infiltration was observed in different groups (Figure 5g & 5h), the secretion of IFN-g by CD4+ T cells and CD8+ T cell was obviously increased in mice after transdermal delivery of FCS/S1/PolyIC compared to that in mice by s.c. injection of S1/polyIC (Figure 5i & 5j), indicating that stronger cytotoxic T cell responses was induced by transdermal delivery of S1 vaccine. Moreover, the percentage of both memory CD4+ and CD8+ T cells in the spleen of mice treated with FCS/S1/polyIC was also dramatically increased on day 90 (Figure S14), while those in mice with subcutaneous administration of S1/PolyIC appeared to be much lower, demonstrating that the transdermal delivery of vaccines would trigger long-term adaptive immune memory effect, which might be resulted from the long retention of co-delivered antigen and adjuvant.
In order to investigate the exact permeation ability of vaccines after transdermal administration, we measured the accumulation of antigens in the lymph node 24 h post transdermal delivery or subcutaneous injection of vaccine using Cy5.5-labeled OVA as the modal antigen. As expected, compared to s.c. injection of OVA, mice after transdermal delivery of vaccine after completely swiping the cream off the skin surface showed lower OVA retention according to the IVIS imaging system (Figure S15). Then we further evaluated the uptake of OVA by DCs in lymph nodes and skin (Figure 5k & 5l). Interestingly, even though the percentage of OVA+ DCs in lymph node of mice with s.c. injection was higher than that of mice with transdermal delivery, the DCs beneath skins showed higher OVA uptake in mice treated by the cream containing FCS/OVA/polyIC, revealing that the transdermal delivery of vaccine could directly activate DCs in situ and the activated DCs could migrate to lymph nodes to trigger further immune responses. In contrast, for the s.c. injection group, antigen and adjuvant would migrate to lymph nodes separately, and were more likely to be depleted by non-specific immune cells and activate DCs with low effectiveness48.
Additionally, we measured the maturation of DC and the activation of CD8+ T cell on one and three days after different treatments. As shown in Figure 5m & 5n, the mice with transdermal delivery of vaccines showed almost similar DC maturation and T cell activation to that of mice with s.c. injection, consistent with the observed T cell activation in the spleen. Therefore, although FCS-based transdermal delivery of vaccine showed less absolute antigen penetration compared to that with s.c. injection, it could trigger almost similar humoral immunity and stronger cell immunity. The additional advantages of such FCS-based transdermal vaccines would be the possibility in self-administration and preferable user compliance. However, further studies such as SARS-CoV-2 virus challenge was not conducted due to our current limitations of experimental conditions in handling viruses.