Biological Effect Tetra-Branched Anti-TNF-Peptide and Coating Ratio-Dependent Penetration of the Peptide-Conjugated Cerium 3 / 4 + Cation-Stabilized Gamma-Maghemite Nanoparticles into Rat Inner Ear after Transtympanic Injection Visualized By MRI

Objective: To evaluate the biological efficacy of TBATP and their distribution in the inner ear after transtympanic injection.


Introduction
Methods: TBATP was synthesized through standard Fmoc solid phase synthesis.A model of TNF-α-induced apoptosis was established in human umbilical vein endothelial cells (HUVECs).The peptides were covalently attached onto CAN-γ-Fe 2 O 3 NPs.Nanoparticle suspension was injected to rat middle ear cavity.Distribution of CAN-γ-Fe 2 O 3 NPs in the inner ear was detected using a 7.0 T MRI machine in combination with Prussian blue staining.
Results: TBATP almost fully suppressed the inhibitory effect on HUVECs induced by TNF-α while the linear one reduced less than half of the effect.CAN-γ-Fe 2 O 3 NPs conjugated to TBATP at a peptide weight ratio of 10% but not 50% efficiently entered the inner ear at 3 h through 2 w post-middle ear administrations and most pronounced at 2 w.
List of Abbreviations: CAN-γ-Fe 2 O 3 NPs: Super-paramagnetic maghemite (γ-Fe 2 O 3 ) nanoparticles using ceric ammonium nitrate (CAN)-mediated oxidation of starting magnetite (Fe 3 O 4 ) nanoparticles; HUVECs: Human umbilical vein endothelial cells; TNF-α: tumor necrosis factor-α autoimmune sensorineural hearing loss [7][8][9].However, antibodies against TNF-α have too large molecular weight to access the therapeutic target.Peptides are smaller in size, more stable during storage, and easier to manipulate than antibodies.To simulate antibody in structure, novel tetra-branched anti-TNF-α peptide selected using phage library with capability of inhibiting human TNF-α binding to its receptors were developed [10].It is clinically relevant to develop a similar therapeutics for treating the inner ear diseases.
The intratympanic delivery of therapeutics is a currently used approach in clinical practice with advantages of avoiding systemic adverse effects and reducing the administration dosage to the individual.In vivo MRI studies demonstrated that intratympanic delivery induced more gadolinium chelate passage into the perimodiolar lymph and lateral wall of mouse cochlea than did the intravenous injection attributed to potential porous structures of medial wall of the scala tympani and spiral ligament extracellular space [11].An efficient passage of agents into the modiolus and lateral wall would raise the possibility that drug delivery to these areas of the cochlea might be favored by intratympanic route.In vivo tracking distribution of therapeutics in the targeting organ is a reliable method to follow the dynamics of the delivered agents.We recently disclosed the preparation of highly hydrophilic anti-aggregative super-paramagnetic maghemite (γ-Fe 2 O 3 ) nanoparticles (NPs) using ceric ammonium nitrate (CAN)-mediated oxidation of starting magnetite (Fe 3 O 4 ) NPs (CAN-γ-Fe 2 O 3 NPs), which were highly stable aqueous suspensions/ferrofluids due to a unique ultrasound-mediated doping process of the Fe 3 O 4 NP surface using lanthanide Ce 3/4+ cations [12].We have also demonstrated that the novel CAN-γ-Fe 2 O 3 NPs is a strong T 2 MRI contrast agent and penetrats both round and oval windows, which has potential application in the molecular imaging of the inner ear [13].
The present study aimed to develop feasible agents for the inner ear therapy as well as diagnosis, and acquire information about their passing through the round and oval windows after conjugation with MRI-traceable nanoparticles.Tetra-branched anti-TNF-α peptides were developed according to a previous report with modifications [10].Biological effect of the peptides in inhibiting TNF-α induced cellular impairment was analyzed in cell cultures.The specific peptides were conjugated to CAN-γ-Fe 2 O 3 NPs at various ratios and the resulting dynamic distributions in the inner ear after middle ear administration have been evaluated in rats using in vivo MRI.

Preparation and Characterization of Tetra-Branched anti-TNF-α-Peptide-Conjugated CAN-γ-Fe 2 O 3 NPs
All the specific chemicals and reagents (analytical grade and/or highest purity level) used in this study, i.e., FeCl

Middle Ear Administration of Tetra-Branched Anti-TNF-α-Peptide-Conjugated CAN-γ-Fe 2 O 3 NPs and MRI of Rat Inner Ear
Four male Sprague Dawley rats (8 ears), weighing between 300 and 350 grams, were maintained in the animal laboratory of A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland.The animals were randomly assigned into 2 groups (NP10, NP-50) (Table 1).The left ear was utilized for the delivery, and the right side was used as a negative control (NC group in Table 1).All the animal experiments have been approved by the Ethical Committee of University of Tampere (permission: ESAVI/3033/04.10.03/2011).Animal care and experimental procedures were conducted in accordance with European legislation.Throughout the experiments, the animals were anesthetized with a 5% isoflurane-oxygen mixture (flow-rate 1.0 L/min) for induction and a 2% isoflurane-oxygen mixture for maintenance via a facemask.Intramuscular injection of enrofloxacin (Baytril®vet, Orion, Turku, Finland) at a dose of 10 mg/kg was applied before the intratympanic administration of NPs to prevent potential infection.The animal's eyes were protected with Viscotears® (Novartis Healthcare A/S, Copenhagen, Denmark).The peptideconjugated CAN-γ-Fe2O3 NPs were administered to the middle ear cavity with an injection tube by touching the surface of the tympanic medial wall according to a previously reported procedure [13,14].A total volume of 10 µL of undiluted CAN-γ-Fe2O3 NP suspension was injected onto the medial wall of the middle ear cavity.The animals remained in the lateral position with the injected ear upward for 15 min before MRI study.After the last MRI measurement, the animals were decapitated for Prussian blue staining for iron in the tissue (Supplementary material 3) (Table 1).

Statistics
In cell culture study, inhibition ratios on cell viability by different treatments were compared using one-way analysis of variance.Values of p<0.05 were accepted as statistically significant.

Characterization of Tetra-Branched Anti-TNF-α Peptide and the Inhibiting Effect on Apoptosis of HUVECs Induced By TNF-α
Purity of both tetra-branched anti-TNF-α and scramble peptides was approved by high performance liquid chromatography that demonstrated the unique specific peak of the peptides (Figure 2).The other sharp peaks might be associated with incomplete cracking of the protecting group for the side chain according to experience.A cell model of apoptosis was successfully established in HUVECs using TNF-α, which showed inhibition on viability of the cells after 24 h incubation in a concentration-dependent response.TNF-α protein at 40 ng/mL induced an average apoptosis of 30.1% of the cells and was selected as the optimized concentration for the following experiments (Figure 3A).Tetra-branched anti-TNF-α peptide suppressed the inhibitory effect on HUVECs induced by TNF-α in a dosage-dependent reaction, and 200 μg/mL of the peptides demonstrated the maximum effect of 69.2% that was used for comparison with other peptides in the following study (Figure 3B).The linear anti-TNF-α peptide also suppressed the inhibitory effect on HUVECs induced by TNF-α but significant lower than did the tetra-branched anti-TNF-α peptide (Figure 3C).Significant morphological changes were detected in rat inner ear at 3 h through 2 w post-middle ear administrations of tetrabranched anti-TNF-α-peptide-conjugated CAN-γ-Fe 2 O 3 NPs at 10% peptide ratio, which demonstrated that area of the ipsilateral cochlea basal turn, vestibule including the ampulla of semicircular canals shrinked in comparison to the contralateral ear (Figure 5).The changes were most pronounced at 2 w after administration, with typical wiping out of the perilymphatic spaces in the basal turn and hook region of the cochlea, and vestibule (Figure 6).This phenomenon was verified by injection of superparamagnetic iron oxide nanoparticles hierarchically coated with oleic acid and Pluronic® F127 copolymers into the scala tympani of rats.The signal in the endolymphatic spaces of the cochlear hook region and vestibule including ampulla became visibly significantly brighter than that in the contralateral side (Figure 6) [15].Similar morphology was visualized in both inner ears of rats receiving middle ear administration of tetra-branched anti-TNF-α-peptide-conjugated CAN-γ-Fe 2 O 3 NPs at 50% peptide ratio.However, no cell population or structure specific distribution of CAN-γ-Fe 2 O 3 NPs conjugated to tetra-branched anti-TNF-α peptide was observed (Figure 7).Inner ear distributions of tetra-branched anti-TNF-α-peptide-conjugated CAN-γ-Fe 2 O 3 NPs were histologically detected using Prussian blue staining (Figure 8 and 9).In accordance with the MRI results, CAN-γ-Fe 2 O 3 NPs conjugated to tetra-branched anti-TNF-α peptide at 50% peptide ratio did not sufficiently enter the inner ear (Figure 9).The present study demonstrated that the tetra-branched anti-TNF-α peptide significantly inhibited apoptosis in HUVECs induced by TNF-α, with an inhibitory effect that was significantly greater than did the linear anti-TNF-α peptide.Both in vivo MRI and histology displayed that CAN-γ-Fe 2 O 3 NPs conjugated to tetra-branched anti-TNF-α peptide at peptide ratio of 10% but not 50% efficiently entered the inner ear and distributed mainly in the perilymphatic spaces after administration in rat middle ear.It indicated that the previously reported novel CAN-γ-Fe 2 O 3 NPs had the capacity to carry the novel tetra-branched anti-TNF-α peptide from middle ear into the inner ear [13].It is possible that the tetra-branched anti-TNF-α peptides are able to pass the middle-inner ear barriers and enter the inner ear by themselves, however, conjugating the peptides onto CAN-γ-Fe 2 O 3 NPs endowed the tetrabranched anti-TNF-α peptide a characteristic of visibility by MRI that will be potentially used as a theranostics agent.

Discussion
In the in vitro study, the tetra-branched anti-TNF-α peptide almost fully suppressed the inhibitory effect on HUVECs induced by TNF-α while the linear anti-TNF-α peptide reduced less than half of the TNF-α effect (Figure 5).The branched anti-TNF-α peptide was proven to inhibit TNF-α binding to human melanoma cells expressing TNF-α receptors.However, protecting effect of the tetra-branched anti-TNF-α peptide on cells against TNF-α receptor signaling activation has not been reported before [10].TNF-αspecific peptide synthesized in linear and tetrameric form showed practically the same binding kinetics to human TNF-α [10].The tetra-branched anti-TNF-α peptide supressed the TNF-α-induced apoptosis in HUVECs more efficiently than did the linear one might be interpreted as following.The TNF-α-specific peptide and TNF-α receptor may bind to human TNF-α at different sites, and the TNF-α-specific peptide-complexed human TNF-α retains the affinity to TNF-α-receptor; the tetra-branched anti-TNF-α peptide has larger spatial volume than the linear one due to the 3-dimentional conformation, and has higher possibility to block the

Figure 1 :
Figure 1: Schema of the tetra-branched peptides MRI studies were performed at the time points of 3 h, 1 d, and 2 w post-middle ear administrations.NC: negative control; NP-10: CAN-γ-Fe 2 O 3 NPs were conjugated to tetra-branched anti-TNF-αpeptide at 10% peptide ratio; NP-50: CAN-γ-Fe 2 O 3 NPs were conjugated to tetra-branched anti-TNF-α peptide at 50% peptide ratio.*The right ear of each animal was used as NC in all studies.

Figure 2 :
Figure 2: High performance liquid chromatography of tetra-branched anti-TNF-α peptide.The unique peak corresponds to either the specific peptides (A) or scramble peptides (B).The other sharp peaks might be associated with incomplete cracking of the protecting group for the side chain

Figure 6 :
Figure 6: Signal changes in rat inner ear on 1 d post-middle ear administration of tetra-branched anti-TNF-α-peptide-conjugated CAN-γ-Fe 2 O 3 NPs at 10% peptide ratio shown by MRI.The narrow strips in the cochlear basal turn and hook region of the delivery ear (L) suggest the scala media (2) while the perilymph signal in the scala vestibuli (1) and scala tympani (3) were wiped out due to distribution of tetrabranched anti-TNF-α-peptide-conjugated CAN-γ-Fe 2 O 3 NPs (A).The remnant endolymph signal in vestibule (Vest) (B), ampulla (Am) of the semicircular canal, and hook region of cochlea (H) (C) became bright.LSCC: Lateral semicircular canal; L: Left; R: Right.Scale bar=1.0mm

Figure 7 :
Figure 7: Insignificant rat inner ear entry of tetra-branched anti-TNF-α-peptide-conjugated CAN-γ-Fe 2 O 3 NPs at 50% peptide ratio shown by MRI.The cochlea and vestibule (Vest) showed symmetric images on both the delivery (L) and contralateral (R) sides at the time points of 3 h (A, B), 1 d (C, D) and 2 w (E, F).Am: ampulla of the semicircular canal; 1 st : basal turn of cochlea; 2 nd : second turn of cochlea; L: Left; R: Right.Scale bar=1.0mm

Table 1 :
Assignments of rat ears in MRI measurements and histology post-middle ear administration of tetra-branched anti-TNF-α-peptide-conjugated CAN-γ-Fe 2 O 3 NPs