Evaluation of the Penetration Process of Fluorescent Collagenase Nanocapsules in a 3D Collagen Gel
AActa
Biomaterialia (2021)
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Evaluation of the penetration process of fluorescent collagenase nanocapsules in a collagen gel Víctor M. Moreno a , b , Alejandro
Baeza c , ∗ , María
Vallet-Regí a , b , ∗ a Dpto.
Química en Ciencias
Farmacéuticas,
Universidad
Complutense de Madrid,
Instituto de Investigación
Sanitaria,
Hospital de Octubre i + Plaza
Ramón y Cajal s/n,
Spain b CIBER de Bioingeniería,
Biomateriales y Nanomedicina,
CIBER-BBN,
Madrid,
Spain c Dpto.
Materiales y Producción
Aeroespacial,
ETSI
Aeronáutica y del Espacio,
Universidad
Politécnica de Madrid,
Madrid,
Spain a r t i c l e i n f o Article history:
Received September
Revised December
Accepted December
Available online December
Keywords:
Nanomedicine
Collagenase nanocapsules
Enzyme release
Polymer nanocapsules a b s t r a c t One of the major limitations of nanomedicine is the scarce penetration of nanoparticles in tumoral tissues. These constrains have been tried to be solved by different strategies, such as the employ of polyethyleneglycol (PEG) to avoid the opsonization or reducing the extracellular matrix (ECM) density. Our research group has developed some strategies to overcome these limitations such as the employ of pH-sensitive collagenase nanocapsules for the digestion of the collagen-rich extracellular matrix present in most of tumoral tissues. However, a deeper understanding of physicochemical kinetics involved in the nanocapsules degrada- tion process is needed to understand the nanocapsule framework degradation process produced during the penetration in the tissue. For this, in this work it has been employed a double-fluorescent labelling strategy of the polymeric enzyme nanocapsule as a crucial chemical tool which allowed the analysis of nanocapsules and free collagenase during the diffusion process throughout a tumour-like collagen ma- trix. This extrinsic label strategy provides far greater advantages for observing biological processes.
For the detection of enzyme, collagenase has been labelled with fluorescein Isothiocyanate (FITC), whereas the nanocapsule surface was labelled with rhodamine
Isothiocyanate (RITC).
Thus, it has been possible to monitor the hydrolysis of nanocapsules and their diffusion throughout a thick Collagen gel during the time, obtaining a detailed temporal evaluation of the pH-sensitive collagenase nanocapsule behaviour. These collagenase nanocapsules displayed a high enzymatic activity in low concentrations at acidic pH, and their efficiency to penetrate into tissue models pave the way to a wide range of possible nanomedical applications, especially in cancer therapy. Statement of significance The present study is focused on the development of a dual fluorescent labelling strategy that allow to monitor the penetration of pH-cleavable polymeric nanocapsules through three- dimensional tissue models. Collagenase was housed within the nanocapsules which were en- gineered to be disassembled at acidic pH leading to enzyme release. In this work, the pH- responsive behaviour of these nanocapsules has been studied employing collagen matrices as tissue models, and the results have confirmed a significant penetration and more homo- geneous distribution of the proteolytic enzymes in mild-acidic conditions. This work presents important applications in the field of cancer therapy and the treatment of fibrotic diseases, among others. © 2020 Published by Elsevier
Ltd on behalf of Acta
Materialia
Inc. Introduction
Nanomedicine has reached great interest in the current decade through the development of nanoparticles and biological nanos- ∗ Corresponding authors.
E-mail addresses: [email protected] (A.
Baeza), [email protected] (M.
Vallet-
Regí). tructures for molecular diagnostics, treatment of cancer and other diseases, and for both applications, as theranostic agents [1] . In the case of cancer therapy, one of the major limitations of the applica- tion of nanomedicines is their scarce penetration in tumours [2 , . This weakness is caused as consequence of the presence of sev- eral well-known biological barriers, that limits the efficacy of the nanomedicine drugs [4] . One of these limitation is the off-target https://doi.org/10.1016/j.actbio.2020.12.022 .M. Moreno, A. Baeza and M. Vallet-Regí Acta
Biomaterialia (2021) accumulation of nanomedicines along the body which can be over- come by anchoring targeting moieties such as peptides or antibod- ies, among others, on nanoparticle surface [5] . Another constrain is the opsonization of the nanotherapeutic by the Mononuclear
Phagocyte
System (MPS) once it is injected in the bloodstream. Plasma proteins are adsorbed onto nanoparticles, forming a protein corona on the surface that undergoes their recognition by unspe- cific receptors of phagocytes, which engulf and destroy the nan- otherapeutics. It has been widely reported that the surface decora- tion with polyethyleneglycol (PEG) [6] reduces this problem hiding the surface to the opsonins present in the blood stream. However, once the nanoparticle reaches the tissue, the existence in most of tumoral tissues of poor lymphatic drainage and dense extracellu- lar matrix (ECM) generates high interstitial fluid pressure that pro- vokes the extravasation of nanotherapeutics to distal regions. Dif- ferent types of nanosystems which present characteristics that par- tially solve some of these limitations have been developed employ- ing an extensive list of materials of different nature: from inorganic systems as metallic [7] and ceramic particles [8 , , organic ones like polymersomes, [10] micelles, [11] liposomes [12] and polymer nanocapsules [13] to hybrid nanodevices which combines both na- tures as protein nanocapsules [14] and even bio-hybrid nanocarri- ers with living organisms as bacteria [15] . The myriad of nanosys- tems developed is endless, but unfortunately, many of them fail in their goal [16] . Our research group has reported some strategies to improve the penetration of nanomedicines in tumoral tissues. One of these strategies is the development of pH-sensitive collagenase nanocap- sules [17] for the digestion of the collagen-rich ECM present in tu- moral tissues, yielding an enhanced penetration of nanoparticles in tumoral tissue models. These collagenase nanocapsules have been used for many clinical applications both in cancer [18] and fibro- sis [19] . The knowledge of physicochemical kinetic involved in the nanocapsules degradation process can help us to better understand the nanocapsule framework degradation process produced during the penetration in the tissue. This knowledge would allow to im- prove the nanocapsule performance for different therapeutic ap- plications. Thus, in this work we describe the development of a fluorescent labelling strategy of the collagenase nanocapsules as a crucial chemical tool that allows the analysis of the diffusion mechanisms across the tissue [20] . This extrinsic labels provide far greater advantages for observing biological processes.
This strat- egy has allowed us to achieve a detailed temporal analysis and evaluation of the collagenase nanocapsule cleavage process to bet- ter understand the free collagenase enzyme diffusion throughout a tumour-like collagen matrix. For the detection of enzyme, we have labelled Collagenase with
Fluorescein
Isothiocyanate (FITC), whereas the nanocapsule surface was labelled with
Rhodamine
Isothiocyanate (RITC). By this way, we could follow during the time the hydrolysis of nanocapsules and the diffusion of collagenase throughout a thick Collagen gel.
This collagen matrix has been widely employed in the literature [21 , as model of extracellular matrix which allows to study the behaviour of nanomedicines in biological tissues. The detailed analysis of the data obtained could clarify some of the factors involved in the process, such as the pen- etration capacity of collagenase in low concentrations, the influ- ence of the pH on this capacity, and the temporal evolution in the penetration reached by collagenase and nanocapsules. Experimental section
Materials
The chemicals were bought to the corresponding supplier and they have been used without further purification. Colla- genase
Type I from Life
Technologies;
Acrylamide (Aa) from
Fluka;
Sodium
Bicarbonate (NaHCO ), Sodium
Phosphate (Na PO ), methacrylate hydrochloride (Am), Ethylene gly- col dimethacrylate (EG),
Ammonium persulfate (APS),
N,N,N’,N’-
Tetramethylethylenediamine (TMEDA),
Fluorescein isothiocyanate isomer I (FITC), Rhodamine B isothiocyanate mixed isomers (RITC), Dimethylsulfoxide (DMSO),
Paraformaldehyde and
Sodium
Hydrox- ide (NaOH) from
Sigma
Aldrich;
Amicon® Ultra-2mL
Centrifugal
Filters
Ultracel®- from
Millipore;
Absolute
Ethanol from
Pan- reac;
Dulbecco’s modified
Eagle’s medium (DMEM),
Fetal
Bovine
Serum (FBS), L -Glutamine and Antibiotic-Antimycotic (Anti-Anti) from
GIBCO.
Rat tail
Collagen (type I) and EnzChek TM Gelati- nase/Collagenase
Assay
Kit from
Life
Technologies;
PBS
Buffer solution pH from Ambion.
Instrumental section:
The hydrodynamic size of protein cap- sules was measured by means of a Zetasizer
Nano ZS (Malvern In- struments) equipped with a nm “red” laser. Transmission
Elec- tron
Microscopy (TEM) was carried out with a JEOL
JEM in- struments operated at equipped with a CCD camera.
Sam- ple preparation was performed by dispersing in distilled water and subsequent deposition onto carbon-coated copper grids. A solution of % of phosphotungstic acid (PTA) pH was employed as stain- ing agent in order to visualize the protein capsules. Fluorescence was measure with
Synergy power supply for Biotek
Laboratory
Instrument
Confocal microscope
Le- ica
SP-2
AOBS with digital camera
Leica
DFC
FX.
Fluorescein-labelling of collagenase (Col-FITC) μL from a solution of FITC in DMSO (1 mg FITC per μl DMSO) were added to a solution of Collagenase (3.1 × − mmol) in mL of NaHCO buffer (0.01 M, pH under stirring. It was stirred during hours at room temperature (RT) and protected from light. After that, the
Collagenase solution was purified by cen- trifugal separation (3.0 rpm, min) with a kDa cut-of filter (AMICON Ultra-2 mL and washed five times with NaHCO buffer (0.01 M pH The enzyme was collected by centrifuga- tion at rpm for min and diluted to a volume of mL with NaHCO buffer (0.01 M pH It was stored at ° C protected from light. Synthesis of fluorescein-collagenase nanocapsules (nCol-F) Previous purified
Fluorescein-Collagenase solution (Col-FITC) was deoxygenated by bubbling N during at least min. Meanwhile,
NaHCO buffer (0.01 M pH was deoxygenated three times with freeze-vacum-N cycles, at room tempera- ture. Then, mmol of acrylamide (Aa), mmol of aminoethylmetacrylate hydrochloride (Am), and mmol of ethyleneglycol dimetacrylate (EG) were dissolved in mL of de- oxygenated NaHCO buffer (0.01 M pH and were added to the solution of Col-FITC.
This mixture was stirred at rpm for min under nitrogen atmosphere at room temperature. Then, mmol of ammonium persulfate (APS) and mmol of N, N, N (cid:3) , N (cid:3) -tetramethyl ethylenediamine (TMEDA) dissolved in mL of the deoxygenated NaHCO buffer (0.01 M pH were added. The re- action mixture was stirred at rpm for min at room tem- perature under inert atmosphere. After this time, the encapsulated enzyme was purified by centrifugal separation with kDa cut-of filters (AMICON Ultra-2 mL KDa) and washed three times with
NaHCO buffer (0.01 M pH These capsules of collagenase were diluted to a mL volume and were preserved at ° C. Rhodamine-labelling of collagenase nanocapsules (nCol-F-Rh) To prepare red fluorescent Collagenase
Nanocapsules, to the previous nanocapsules solution (nCol-F) was added μL from a .M. Moreno, A. Baeza and M. Vallet-Regí Acta
Biomaterialia (2021) solution of RITC in DMSO (1 mg RITC per μL DMSO). It was stirred during hours at RT and protected from light. After that, the nanocapsules solution was purified by centrifugal separation (3.0 rpm, min) with a kDa cut-of filter (AMICON Ultra- mL and washed five times with NaHCO buffer (0.01 M pH The enzyme was collected by centrifugation at rpm for min and diluted to a volume of mL with NaHCO buffer (0.01 M pH It was stored at ° C. Protein concentration was measured following the
Bicinchoninic
Assay (BCA) protocol for
Pro- tein quantification [23] . For this, μL of the protein sample were added to μL of a working solution containing Bicinchoninic acid and
Cupric
Sulphate.
Mixture was incubated for min at ° C and then absorbance was measured at nm. The green and red fluorescence of nCol-F-Rh was determined at λ abs / λ em of nm/520 nm for FITC and λ abs / λ em of nm/576 nm for RITC, respectively.
Enzymatic activity measurements
The enzymatic activity of native collagenase (Col), Col-FITC, nCol-F and nCol-F-Rh was evaluated using and following the proto- col
EnzChek
Gelatinase/Collagenase
Assay
Kit.
For this experiment, μ L of Phosphate buffer μ L of Collagen-FITC, and μ L of each collagenase sample were used. All samples were standard- ized to an enzymatic activity of U/mL in PBS solution at pH at room temperature and their enzymatic activity was stud- ied at different times every min, measuring the accumulated green fluorescence intensity in a fluorescence microplate reader ( λ abs = nm / λ em = nm). Enzymatic activity of Col and nCol-F-Rh samples tested at pH and ° C was measured as described, but samples were in this case standardized to an enzymatic activity of U/mL in PBS solution.
Preparation of collagen gels Briefly, ml of Rat
Tail
Collagen type I (3 mg • ml − ) and mL of complemented DMEM (DMEM medium with % FBS and L - Glutamine) were mixed at ° C and subsequently μL of a M NaOH solution was added until neutral pH.
Then, ml of FBS and ml of PBS were added to the solution at ° C. Afterwards, mL of this mixture was added to the wells of a plate and incubated at ° C at an atmosphere of % CO for h to promote the collagen gelification. To avoid gel cracking, the edge of each well was encircled with a needle. Finally, μL of PBS was added, and incubated at ° C at an atmosphere of % CO overnight. The resulting collagen gels were employed for the further experiments one day after gels formation.
Nanocapsules penetration evaluation in collagen gels at h end-point experiment To the previously prepared collagen gels, the supernatant was removed and μL of a mg/mL nanocapsules solution (theoretical activity of U/mL) in M Na PO pH buffer and M Na PO pH buffer, respectively, was added in the centre of the gel. Next day, μL of a % Glutaraldehyde solu- tion in PBS was added for
Then, gels were washed with PBS and analysed by Confocal microscopy at different depths. Temporal evaluation of nanocapsules penetration in collagen gels during h According to the protocol described in Section , gels were prepared with the same procedure. However, in this case mL of the obtained mixture was added to a four wells Chamber
Slide TM system, then was delicately removed μL of mixture from each well and it was incubated at ° C at an atmosphere of % CO overnight. The resulting collagen gels were employed one day after gels formation.
Next day, a solution of nanocapsules with a pro- tein concentration (calculated according to protocol described in section) of mg/mL (corresponds to a theoretical activity of U/mL) was incubated in different conditions to obtain three samples: M Na PO pH buffer at ° C for nCol-A, in M Na PO pH buffer for nCol-B , and in M Na PO pH buffer for nCol-C. nCol-A was incubated in a lab oven at ° C for h to get the enzyme inactivated by thermal shock, while nCol- B and nCol-C were kept the same time at ° C to preserve their enzymatic activity. Then, a μL of each sample was added on top of these gels, and red and green fluorescence were monitored at different depths in h intervals during h at ° C by confocal microscopy. Statistical analysis
The results shown throughout the article are displayed as mean ± standard error of the mean (SEM), unless otherwise stated. Sta- tistical evaluation of quantitative data was carried out using the Student’s
T-test. p values < were considered to be significant. Results and discussion
Labelling of collagenase enzyme with FITC (Col-FITC)
For the correct identification of Collagenase distribution and diffusion throughout a tissue, it is necessary to label the enzyme with an intense fluorophore. It is known by proteomic studies that Collagenase, as most of enzymes, possess several Lysine groups on its tertiary structure. The
Fluorescein-labelled
Collagenase (Col-
FITC) was obtained through the covalent anchoring of the fluores- cent dye to the amine groups from Lysine amino acids presents in the enzyme. For this,
Fluorescein
Isothiocyanate (FITC) dissolved in DMSO was added to a solution of Collagenase in NaHCO buffer pH It was stirred during hours at RT to originate the car- bamate covalent attachment by nucleophilic attack of free amine groups from lysines present in the enzyme to the isothiocyanate group of the fluorophore. The carbamate displays high chemical and proteolytic stability in physiological conditions due to the low electrophilicity of carbonyl group [24] . Finally, the obtained
FITC- labelled
Collagenase was incorporated into a kDa cut-of filter (Amicon) and was centrifugated and washed several times with buffer to remove the unreacted FITC. By this way, the Col-FITC re- mains retained in the filter after centrifugation cycles at rpm for min, while the washing solutions sweep along the unreacted FITC.
The green fluorescence was measured after each washing step requiring five washing steps to remove completely the excess of FITC.
Then, the labelled protein was collected at rpm for min. The variation in the surface charge of protein was tested by measuring the Z potential of Col-FITC, which gave a value of – 30.2 mV. Comparing this value with native collagenase (- mV) indi- cates that the incorporation of FITC reduce the amine groups in the surface of the protein and therefore the value become more neg- ative. The correct anchoring of FITC to collagenase was also con- firmed in further steps measuring the fluorescence intensity of the nanocapsules. Synthesis of fluorescein-collagenase nanocapsules (nCol-F) The incorporation of fluorophores on the nanocapsule surface allows us to monitor their behavior inside the host tissue. The for- mation of a polymeric nanocapsule around the collagenase pre- serve it from degradation and therefore, it maintains its enzymatic .M. Moreno, A. Baeza and M. Vallet-Regí Acta
Biomaterialia (2021)
Fig. A) Schematic synthetic procedure of collagenase nanocapsules. B) DLS analysis of free collagenase and nCol-F nanocapsules. C) TEM micrography of nCol-F, showing an average diameter of nm. activity. For the encapsulation of Col-FITC is mandatory to perform the polymerization of monomers in oxygen-free conditions, to pre- vent the quenching of formed free radicals with the O dissolved in the buffer. For this reason, the solution containing
Col-FITC was deoxygenated by bubbling N . The buffer was also deoxygenated with three freeze-thaw cycles under N atmosphere to ensure the removal of oxygen in the aqueous buffer. The monomers employed for the nanocapsule formation were acrylamide (Aa) as a structural monomer to complete the whole polymerization and hydrochloride (Am) as an amine groups provider monomer. Amino groups provide re- active functional groups for the anchoring of the corresponding flu- orophores at the same time that to provide colloidal stability to the nanocapsules in the ionic aqueous medium. Finally, ethyleneglycol dimetacrylate (EG) was employed as pH-sensitive cross-linker, al- lowing to the capsule to be hydrolysed when the pH is acid, as it has been reported previously [18] . These monomers were dissolved in previously deoxygenated NaHCO buffer and were subsequently added to the deoxygenated solution of Col-FITC.
This mixture was stirred under N atmosphere to get the protein surrounded by monomers. Then, the slowly addition of radical initiators APS and
TMEDA initiates the polymerization between monomers which are surrounding the protein.
This reaction mixture was stirred at RT under inert atmosphere to complete the polymerization process around the protein yielding the nanocapsules. After this, encapsu- lated enzymes were purified by centrifugation employing kDa cut-of filters (Amicon) and then, they were washed three times with NaHCO buffer to remove the excess of monomers yield- ing collagenase nanocapsules (nCol-F). A schematic procedure of this synthesis is shown in Fig. -A . The hydrodynamic diameter of the nanocapsules was measured by Dynamic
Light
Scattering (DLS), and their size was compared with collagenase before encap- sulation.
Collagenase shown an average diameter of nm, while nCol-F shown a distribution centred in nm ( Fig. -B) . This size is consistent with the average size obtained by TEM analysis of around nm ( Fig. -C ). Zeta potential measurement changed from – 30.2 mV of Col-FITC to a value of – 14.1 mV, which became less negative due to the presence of amine groups in the nanocap- sules surface. Rhodamine-labelling of nCol-F (nCol-F-Rh) and nanocapsules activity measurements For the study of nanocapsules hydrolysis process, collagenase was labelled with FITC for the correct protein identification.
These
Col-FITC were encapsulated, and the obtained nanocapsules (nCol- F) were labelled with another fluorophore for their visualiza- tion by fluorescence microscopy throughout the gel. The labelling mechanism is the same as mentioned before; the correspondent fluorophore-isothiocyanate react with amine groups from the poly- meric framework of the nanocapsules, yielding a stable carbamate conjugate. For our purpose, the nanocapsules were labelled with .M.
Moreno, A. Baeza and M. Vallet-Regí Acta
Biomaterialia (2021)
Fig. Left:
Schematic procedure for the labelling of Collagenase nanocapsules with
FITC,
RITC and
Cy7.
Right:
Image of a control Collagenase nanocapsules and
Fluorophores- labelled
Collagenase nanocapsules.
Fig. Schematic representation of double-fluorescent labelling steps of Collagenase nanocapsules.
Rhodamine
Isothiocyanate (RITC), which provides a red fluores- cence to the nanocapsules without interference with the green flu- orescence delivered by Col-FITC.
However, it is noteworthy that other fluorophores can be used such as FITC or Cyanine7-NHS ester (Cy7). In three cases, the fluorescence was kept after several wash- ing steps, as is showed in Fig. . These fluorophores could be used indistinctly, depending on the desired wavelength. Dual red/green fluorescence in Collagenase nanocapsules (nCol-
F-Rh) were synthesized employing
RITC as fluorophore following a similar procedure to the employed in FITC grafting.
After that, the obtained nCol-F-Rh were purified by centrifugation employ- ing kDa Amicon following the same steps carried out in the first labelling to remove the excess of RITC. A short scheme of the double-labelling process is shown in Fig. . The Z potential of nCol- F-Rh was measured, and the value increased from – 14.1 mV of nCol-F to – 7.8 mV. This change in the surface charge is consistent with the incorporation of RITC group on the surface of nanocap- sules, which is positively charged at pH of The encapsulation of collagenase should reduce its enzymatic activity because the encapsulated enzyme cannot access to the substrate (collagen) due to the presence of the polymeric shell. It is necessary to release the enzyme from the polymeric nanocapsule to restore its capacity to digest the collagen matrix. The transient enzymatic activity loss of the encapsulated collagenase was deter- mined measuring the proteolytic capacity of nanocapsules in com- parison with free collagenase which suffered the same purification steps but without being encapsulated within a polymeric shell. The enzymatic activity of the collagenase trapped in the collagenase nanocapsules can be reduced due to the friction forces and tem- perature increases required in the numerous purification steps re- quired in the synthesis and this reduction should be discarded to the reduction produced by the encapsulation process ( Fig. A).
In- terestingly, the enzymatic activity of free collagenase was rapidly reduced in mild acidic conditions whereas the encapsulated col- lagenase maintained its proteolytic capacity during longer times. Thus, the encapsulated enzyme retained more than % of enzy- matic activity after hours at pH = in comparison with free enzyme which showed negligible activity ( Fig. B). In this graphic, the enzymatic activity of free collagenase was normalized to the corresponding % enzyme activity in nanocapsule form with a correction factor of to avoid the overestimation of the enzy- matic activity of free collagenase. It is noticeable that the decrease of enzymatic activity during nanocapsules formation and labelling of fluorophores processes is around a % comparing with the activity of native collagenase. This drastic reduction was not permanent while it was recovered once the nanocapsules were hydrolysed as it is showed in Fig. B. The polymeric nanocapsule acts as a shield that protect the en- zyme but also cover the active site. It justifies the high decrease in the enzymatic activity when the enzyme is encapsulated. Nev- ertheless, as it was reported in previous works, once the nanocap- sule is degraded by the effect of the pH, the enzymatic activity is mainly recovered [18] . Both samples were stored at ° C dur- ing h and their activity was newly measured, but there are no significant change in their enzymatic activities. Free enzyme and nCol-F-Rh preserves their activity at low temperatures. It is also important to ensure that the fluorescence capacity of fluorescein and rhodamine is not affected significatively during the synthesis process. For this reason, it was measured the red and green fluorescence emitted from nCol-F-Rh, and data obtained were standardized to mg of nanocapsules. For green fluorescence was obtained a concentration value of μg of FITC in mg of pro- tein. In the case of Red
Fluorescence of nCol-F-Rh, it was obtained a value of μg of RITC in mg of protein . This value is around .M. Moreno, A. Baeza and M. Vallet-Regí Acta
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Fig. A) Enzymatic activity after each synthetic step, B) Enzymatic activity of collagenase nanocapsules and collagenase at mild acidic conditions. higher than the fluorescence derivate from FITC, due to the high availability of amine groups in the surface of the nanocap- sules comparing with the amine groups present in the enzyme. Evaluation of the penetration of nCol-F-Rh nanocapsules in collagen gels In order to estimate the capacity of collagenase to penetrate into collagen gels designed to mimic an extracellular matrix, nCol- F-Rh were incubated in a three-dimensional Collagen gel at differ- ent pH to promote the hydrolysis of the pH sensitive red-labelled polymeric shell of nanocapsules and the subsequent diffusion of Col-FITC enzyme retained inside due to the continuous digestion of the collagen from the gel. Both end-point penetration experi- ment and temporal evaluation penetration were performed for the evaluation of colour differences inside the gels over time. Nanocapsules penetration evaluation in a h end-point experiment One day after the formation of gels, the resulting collagen gels were employed for the nCol-F-Rh penetration experiment. For this purpose, two samples of nanocapsules solution (2,57 U/mL) were prepared at pH and pH Then, they were added on the centre of the collagen gel and they were incubated for h. Next day, gels were fixed with a % Glutaraldehyde solution in PBS to stop the collagen degradation and to protect the fluores- cence capacity of fluorophores. Then, gels were washed with
PBS and analysed by Confocal microscopy at different depths, obtaining the XY planes superposition in the Z-axis for different sam ples at physiological pH, mild-acidic conditions and gel without nCol-F-Rh as control. The obtained representations can be observed in Fig. . Due to the pH-sensitivity nature of the collagenase nanocapsules, at pH the hydrolyzation of the pH-sensitive monomers present in the polymeric shell was faster than the observed at physiological pH. Mild-acidic conditions provoked rapid release of the Col-FITC from nanocapsules and allowed to the enzyme to digest the colla- gen matrix of the gel. As result of this, higher penetration rate was observed at acid pH in comparison with physiological pH. This pro- cess is schematized in Fig. -A. Collagenase labelled with
FITC were more homogeneously distributed in the entire gel reaching deeper zones in mild acidic conditions than in the case of nanocapsules incubated at physiological pH ( Fig. C and D). It is observable that red fluorescence (represented in purple) derived from the labelling of the polymeric shell of nanocapsules was slightly more abundant inside the gel in the sample at pH in comparison with pH The triggered release of Col-FITC at acid pH allowed a fast diges- tion of collagen from gel and therefore, nanocapsules were capa- ble to penetrate deeper in the gel than the nanocapsules exposed to physiological pH. The control gel did not show autofluorescence derived from the collagen ( Fig. B).
Temporal evaluation of nCol-F-Rh nanocapsules penetration in collagen gels for hours For a precise temporal analysis of the nanocapsules hydrolysis progress at physiological and acidic pH, the experiment was per- formed in a four wells Chamber
Slide TM system, which was then coupled to a confocal microscope for gel analysis. Gels were pre- pared, as described in Section , inside this Chamber slide.
Then, it was inserted in a humid chamber to prevent the dehydration of the gels (thereby avoiding the loss of its volume) and the tem- perature was kept at ° C for an optimal enzyme activity and also to assure the same environmental conditions between sam- ples. This assembly allowed us to analyse sequentially the samples every hours for a depth of μm in each gel during a period of hours. For this, three samples were incubated during h in different conditions. One of them correspond to inactive nCol-F-Rh ( nCol-A ) which was incubated at ° C and pH to eliminate the enzymatic activity by thermal denaturalization. Other sample was nCol-F-Rh incubated at pH ( nCol-B ) to emulate physiological conditions. Finally, the last sample was nCol-F-Rh incubated at pH ( nCol-C ). μL of each sample were added on top of gels, and then, red and green fluorescence were monitored for each sample. Gels were analysed and the projected fluorescence signals in the ZY plane were represented to appreciate the nanocapsule and col- lagenase penetration in each case. The penetration depth of each gel each h of analysis can be observed in the videos from Sup- porting
Information (S1 for nCol-A; S2 for nCol-B and S3 for nCol- C).
The comparative analysis between nCol-F-Rh at pH and pH in Fig. -A show that red fluorescence was present in the up- per layers of the gel at both physiological and acidic pH after h of experiment. However, in the case of green fluorescence in Fig. -B, it is observable that after h of experiment, the fluores- cence front reached deeper regions of the gel at pH comparing with pH This fact suggests that nanocapsules exposed to mild acidic conditions were hydrolysed in higher amount than the ones exposed to physiological pH and therefore, they release higher amount of collagenase to the media which were able to diffuse to .M. Moreno, A. Baeza and M. Vallet-Regí Acta
Biomaterialia (2021)
Fig. A) Schematic representation of nCol-F-Rh degradation inside the collagen gel. B) Control
Collagen gel without nCol-F-Rh. C) and D) Confocal images
Z-axis superpo- sition of Collagen gel incubated with nCol-F-Rh for h at physiological pH (C) and at Acid pH (D) for upper and lower fronts of the gel. Observation depth in each case were μ m for C and μ m for D, respectively. Images were taken each μ m. Scale bar: μm. .M. Moreno, A. Baeza and M. Vallet-Regí Acta
Biomaterialia (2021)
Fig. Comparative confocal images of a Z-axis projection of collagen gel incubated with nCol-F-Rh at physiological pH (Left images) and acidic pH (Right images) at times and h. A) Images correspond with
Red fluorescence related to RITC . B) Images correspond with
Green fluorescence of FITC.
All gels were compared for a depth of μm. inner zones of the gel. Interestingly, polymeric nanocapsules could not penetrate deeply into the tissue even in the case of the ones exposed to mild acidic conditions. Polymeric nanocapsules main- tain their integrity due to the presence of the pH-sensitive cross- linkers which could be not completely hydrolysed in this period of time, even in the case of mild-acidic conditions. Therefore, the hydrolysis of the polymeric framework in this last condition is enough to release the collagenase but not enough to allow a free diffusion of the hydrolysed polymer chains into the gel. At physiological pH of the temporal progression of green fluorescence across the inner gel is slower comparing with the pro- gression in the case of enzyme previously incubated at pH of In this case, the distribution of the fluorescent “cloud” is much wider along the gel and comparatively higher than the case of pH indicating that the premature hydrolysis of the nanocap- sules allowed the collagenase to diffuse freely inside the Collagen gel and thereby, digested the collagen achieving higher penetration rates in comparison with the nanocapsules incubated at physiolog- ical pH. To obtain a detailed profile for the evolution of red and green fluorescence along the gel, the raw fluorescence data of nCol-F- Rh in different conditions ( nCol-A, B and C ) was extracted and analysed. The green fluorescence data were represented in an Area graph, overlaying all the Z planes of the gel to obtain a Z projection of the whole gel. In that way, it is represented the fluorescence in- tensity area vs gel depth for each time. Each time of analysis are overlaid in the graph to appreciate the progress of depth vs time in the sample. Analysing the green fluorescence (relative to Col-
FITC) for the case of nCol-A ( Fig. -A), it was observable that the fluorescence intensity was concentrated in first μm of the gel, and the intensity profile did not change over time. This ineffective penetration indicated that the nanocapsules have lost their enzy- .M.
Moreno, A. Baeza and M. Vallet-Regí Acta
Biomaterialia (2021)
Fig. Green
Fluorescence intensity histograms for each gel layer analysed every hours. A) Histogram correspondent to inactivated nCol-F-Rh ( nCol-A ). B) Histogram correspondent to nCol-F-Rh preincubated at pH ( nCol-B ). C) Histogram correspondent to nCol-F-Rh preincubated at pH ( nCol-C ). .M. Moreno, A. Baeza and M. Vallet-Regí Acta
Biomaterialia (2021)
Fig. Red
Fluorescence intensity histograms for each gel layer analysed every hours. A) Histogram correspondent to inactivated nCol-F-Rh ( nCol-A ). B) Histogram corre- spondent to nCol-F-Rh preincubated at pH ( nCol-B ). C) Histogram correspondent to nCol-F-Rh preincubated at pH ( nCol-C ). .M. Moreno, A. Baeza and M. Vallet-Regí Acta
Biomaterialia (2021) matic activity with the thermal shock and there was no digestion of the collagen. In the case of nCol-B sample ( Fig. -B) , it was ob- servable that the fluorescence intensity profile has slightly progress comparing with inactive enzyme, reaching a fluorescence front be- tween μm depth at h of analysis, with a poor increase of the depth at the time of h. This effect can be attributed to a very slow degradation of nanocapsules at pH which provoked a poor collagen degradation, and consequently, poor penetration of fluorescent collagenase. Finally, in nCol-C sample ( Fig. -C), it is noteworthy that the fluorescence intensity profile has considerably advanced to deeper zones in the gel in comparison with nanocap- sules at pH In this case, at h of experiment the whole flu- orescence front was situated between μm depth of the gel. Then, the front advances over time, achieving a wide distribution of the fluorescence between and μm depth after h of anal- ysis. The faster degradation of nanocapsules at pH allowed the fluorescent enzyme to rapidly degrade the collagen, obtaining two- fold enzyme penetration ratios for the same time, and with very low enzyme concentrations. Through the analysis of the histograms for red fluorescence in- tensity (relative to nanocapsules polymeric structure) it was ob- served that the fluorescence intensity was limited to the first μm of the collagen gel with nCol-A ( Fig. A) whereas in the case of nCol-B ( Fig. B) and nCol-C ( Fig. C) only a little fraction of poly- meric nanocapsules were able to penetrate deeply into the gel: μm at physiological pH and μm, in the case of nanocap- sules incubated in mild-acidic conditions. Most of the red fluores- cence dots were concentrated in the first μm of the gel in both cases. These results suggest that nanocapsules did not penetrate by themselves remaining in the close surface of the gel. The reason of this poor penetration can be that, even in the case of nanocapsules exposed to mild acidic conditions, the nanocapsules retained a cer- tain amount of non-hydrolysed crosslinkers which partially main- tained their integrity and therefore, their penetration into the gel were compromised. As result of this fact, only a low amount of red fluorescent dots (which correspond to unaltered or hydrolysed nanocapsules) were detected in deep zones of the gel. In any case, the presence of these red dots in deep zones of the gel are higher in the case of nanocapsules exposed to mild acidic conditions due to the higher release of collagenase in the zone. This also reinforce the idea that nanocapsules present low activity before degrada- tion due to the presence of polymer shell that act as a “shield”which protect the enzyme, as has been mentioned above. How- ever, the premature degradation of nanocapsules at pH release the enzyme from the polymeric framework, recovering the enzy- matic activity and reaching higher penetration ratios of green flo- rescence dots due to the presence of FITC-labelled enzyme.
Thus, the nanocapsules acted as collagenase reservoir that, at mild acidic conditions, released collagenase which can diffuse to deep zones of the tissue. Conclusions In conclusion, a new strategy to evaluate the hydrolysis and penetration of pH-sensitive collagenase nanocapsules is presented. For this, the collagenase nanocapsules were labelled with two dif- ferent fluorophores, fluorescein and rhodamine.
Fluorescein was attached to native collagenase enzyme while rhodamine was an- chored on the nanocapsule surface. The enzyme activity was al- tered in the fluorophore anchoring and polymer coating processes but did not permanently compromise its functionality. The double labelling allowed us to evaluate the penetration capacity of colla- genase nanocapsules, and additionally the diffusion capacity of col- lagenase enzyme once the polymeric framework of nanocapsules was hydrolysed. Due to automatic-confocal microscopy analysis it was assured a complete reproducibility of procedure conditions be- tween samples. As a result, it was observed that Col-FITC diffusion capacity in acidic pH was higher than in the case of physiological pH, due to the pH-responsiveness nature of these nanocapsules. It was also analysed in detail the evolution of fluorescence over time inside the gel, obtaining that the enzyme was capable to penetrate two-fold deeper in acidic medium than in physiological medium in hours, even though the enzyme concentration was low. Mean- while, the red fluorescence of polymeric shell was retained in both cases in the first few layers and did not penetrated into the tissue. These collagenase nanocapsules displayed a high enzymatic activ- ity at low concentrations and their optimal operation opens a wide range of possible nanomedical applications. Supporting
Information
Video of the penetration of nCol-F-Rh at pH and in Col- lagen gels.
Declaration of Competing
Interest
The authors declare that they have no known competing finan- cial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments
Author
Contributions:
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest. This work was supported by European
Research
Council;
ERC-2015-AdG (VERDI),
Proposal
No. and by ERC- (N ° DECOMPACT.
The authors wish to thank the ICTS
Centro
Nacional de Micro- scopia
Electrónica (Spain) and
CAI
Cytometer and
Fluorescence mi- croscopy of the Universidad
Complutense de Madrid (Spain) for the assistance and
Servier
Medical art for the
Creative
Commons fig- ures.
Supplementary materials
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