**2. Amyloid aggregation and dendrimers**

According to the amyloid cascade hypothesis, Aβ peptides are important players triggering the AD development. Multiple in vitro studies have demonstrated that the Aβ peptides can form fibrils and other aggregates called oligomers. The formation of insoluble Aβ fibril follows a nucleation-dependent polymerization mechanism (**Figure 2**) as described [29]. The formation of soluble Aβ oligomers in vivo is largely unknown; it is believed that soluble Aβ oligomers may precede fibril formation [30] and are more toxic than mature Aβ fibrils [31].

In the search for drugs that would inhibit neuronal death in Alzheimer's disease, one of the ways one can use is to find compounds that interfere with Aβ, cleaning the brain tissues from neurotoxic Aβ oligomers. It has been demonstrated that PPI dendrimers modified with maltose are capable of interfering with the amyloid formation in vitro [18, 28, 32, 33]*.* Amyloid fibril formation is usually monitored

#### **Figure 2.**

*Example of amyloid fibrils and amyloid oligomers. (A) Electron micrographs of the Aβ(1–40) fibrils (B) Aβ(1–40) oligomers prepared as described [9]. Scale bar is 200 nm.*

**93**

*Glycodendrimers as Potential Multitalented Therapeutics in Alzheimer's Disease*

using the fluorescence dye thioflavin T (ThT). The dye becomes fluorescent when interacting with the ordered β-sheet structures characteristic for amyloid fibrils. With the fibril growth, the ThT fluorescence increases until its value reaches a plateau. **Figure 3** demonstrates the sigmoid-shaped line corresponding to the ThT kinetics corresponding to the fibril growth of Aβ(1–40), where the lag (nucleation) phase is followed by the elongation phase and plateau; when all ThT molecules have intercalated into β-sheets of the amyloid fibrils, the aggregation kinetics of

*Characteristic aggregation curve for amyloid fibril formation. Sigmoid-shaped curve 5 μM recombinant Aβ(1–42) kinetics as detected by ThT fluorescence over time and displayed as % of total ThT binding. Area (A) corresponds to the lag phase (nucleation), area (B) corresponds to the growing phase, and area (C) corresponds* 

*to final ThT fluorescence plateau. Inset: molecular structure of ThT.*

PPI dendrimers modified with maltose may, in the case of Aβ(1–40) or Aβ(1–42),

interfere with amyloid fibril formation in a concentration-dependent manner, indicating that maltose PPI dendrimers bind amyloid proteins [18]. **Figure 4** demonstrates the ThT fluorescent kinetics of Aβ(1–40) and Aβ(1–42) in the presence of maltose PPI dendrimers. As expected, Aβ alone forms the typical amyloid fibrils [30]. However, when the maltose PPI dendrimers are present, the morphology of amyloid fibrils is altered, demonstrating binding of the dendrimers to Aβ [18, 28, 35, 36]. The electron micrograph shows the morphology of amyloid fibril in the presence of maltose PPI dendrimers. Fibril clumps were generated by incubating maltose PPI dendrimers with Aβ(1–40). As it has been suggested that dendrimers interact with Aβ thus, fibrils seem to be varnished by maltose dendrimers and clumped together, and importantly, no Aβ oligomers were observed in the presence of maltose PPI dendrimers [18]. Thus it is reasonable to think that maltose dendrimers interacting with Aβ may form hybrid fibrils, shifting the balance between

oligomeric and fibrillar forms of Aβ toward less toxic hybrid products.

biological applications [37]. It was observed that unmodified PPI dendrimers have high intrinsic toxicity for cells [38, 39]. It was hypothesized that this toxicity could be related to the dendrimer capacity of establishing strong interactions of electrostatic nature [40]. It has been demonstrated that dendrimers with a surface decorated by polysaccharides, such as maltose or maltotriose, confer less toxicity [41, 42]. The charge of the dendrimer covered by polysaccharides is close to neutral;

Dendrimers' intrinsic toxicity is an important issue in relation to their potential

*DOI: http://dx.doi.org/10.5772/intechopen.88974*

amyloids is reviewed [34].

**Figure 3.**

*Glycodendrimers as Potential Multitalented Therapeutics in Alzheimer's Disease DOI: http://dx.doi.org/10.5772/intechopen.88974*

#### **Figure 3.**

*Neuroprotection - New Approaches and Prospects*

research and possibly treatment of brain diseases.

**2. Amyloid aggregation and dendrimers**

tion [30] and are more toxic than mature Aβ fibrils [31].

second generation have 16 functional groups on their surface, the third generation has 32, and the fourth dendrimer generation has 64 functional groups. Strikingly, the number of terminal groups increases exponentially, while the size increases linearly. The terminal groups on the dendrimer surface can be used for surface modification and dendrimer functionalization. Such modifications could change dendrimers' surface charge and, for example, reduce toxicity associated with a cationic surface charge as reviewed by Appelhans et al. [23]. Dendrimers are most commonly synthesized using divergent or convergent different synthetic pathways [24]. Importantly, the high tunability of dendrimers' surface allows endless possibilities for dendrimers' biomedical applications, for example, for pharmaceutical applications, the terminal groups can be functionalized with different active conjugates such as specifically targeting antibodies, drugs, metal ions or imaging agents, and more [25]. Moreover, several research groups demonstrated that some types of dendrimers are able to cross the BBB [22, 26–28], showing their applicability for the

In the present chapter, I summarize the experimental evidence showing that functionalized poly(propylene imine) dendrimers may provide multitargeting properties for dendrimers increasing their potential for the treatment of AD.

According to the amyloid cascade hypothesis, Aβ peptides are important players triggering the AD development. Multiple in vitro studies have demonstrated that the Aβ peptides can form fibrils and other aggregates called oligomers. The formation of insoluble Aβ fibril follows a nucleation-dependent polymerization mechanism (**Figure 2**) as described [29]. The formation of soluble Aβ oligomers in vivo is largely unknown; it is believed that soluble Aβ oligomers may precede fibril forma-

In the search for drugs that would inhibit neuronal death in Alzheimer's disease, one of the ways one can use is to find compounds that interfere with Aβ, cleaning the brain tissues from neurotoxic Aβ oligomers. It has been demonstrated that PPI dendrimers modified with maltose are capable of interfering with the amyloid formation in vitro [18, 28, 32, 33]*.* Amyloid fibril formation is usually monitored

*Example of amyloid fibrils and amyloid oligomers. (A) Electron micrographs of the Aβ(1–40) fibrils* 

*(B) Aβ(1–40) oligomers prepared as described [9]. Scale bar is 200 nm.*

**92**

**Figure 2.**

*Characteristic aggregation curve for amyloid fibril formation. Sigmoid-shaped curve 5 μM recombinant Aβ(1–42) kinetics as detected by ThT fluorescence over time and displayed as % of total ThT binding. Area (A) corresponds to the lag phase (nucleation), area (B) corresponds to the growing phase, and area (C) corresponds to final ThT fluorescence plateau. Inset: molecular structure of ThT.*

using the fluorescence dye thioflavin T (ThT). The dye becomes fluorescent when interacting with the ordered β-sheet structures characteristic for amyloid fibrils. With the fibril growth, the ThT fluorescence increases until its value reaches a plateau. **Figure 3** demonstrates the sigmoid-shaped line corresponding to the ThT kinetics corresponding to the fibril growth of Aβ(1–40), where the lag (nucleation) phase is followed by the elongation phase and plateau; when all ThT molecules have intercalated into β-sheets of the amyloid fibrils, the aggregation kinetics of amyloids is reviewed [34].

PPI dendrimers modified with maltose may, in the case of Aβ(1–40) or Aβ(1–42), interfere with amyloid fibril formation in a concentration-dependent manner, indicating that maltose PPI dendrimers bind amyloid proteins [18]. **Figure 4** demonstrates the ThT fluorescent kinetics of Aβ(1–40) and Aβ(1–42) in the presence of maltose PPI dendrimers. As expected, Aβ alone forms the typical amyloid fibrils [30]. However, when the maltose PPI dendrimers are present, the morphology of amyloid fibrils is altered, demonstrating binding of the dendrimers to Aβ [18, 28, 35, 36]. The electron micrograph shows the morphology of amyloid fibril in the presence of maltose PPI dendrimers. Fibril clumps were generated by incubating maltose PPI dendrimers with Aβ(1–40). As it has been suggested that dendrimers interact with Aβ thus, fibrils seem to be varnished by maltose dendrimers and clumped together, and importantly, no Aβ oligomers were observed in the presence of maltose PPI dendrimers [18]. Thus it is reasonable to think that maltose dendrimers interacting with Aβ may form hybrid fibrils, shifting the balance between oligomeric and fibrillar forms of Aβ toward less toxic hybrid products.

Dendrimers' intrinsic toxicity is an important issue in relation to their potential biological applications [37]. It was observed that unmodified PPI dendrimers have high intrinsic toxicity for cells [38, 39]. It was hypothesized that this toxicity could be related to the dendrimer capacity of establishing strong interactions of electrostatic nature [40]. It has been demonstrated that dendrimers with a surface decorated by polysaccharides, such as maltose or maltotriose, confer less toxicity [41, 42]. The charge of the dendrimer covered by polysaccharides is close to neutral;

**Figure 4.**

*Effect of G4 histidine-maltose PPI dendrimers on the fibrillization of Aβ. (A) Aggregation of 20 μM Aβ(1–40) in the absence (red) and the presence of histidine-maltose PPI dendrimers. (Magenta) 20 μM Aβ(1–40) in the presence of dendrimers at dendrimer/peptide ratio = 0.1, (blue) 20 μM Aβ(1–40) in the presence of dendrimers at dendrimer/peptide ratio = 1. (B) Aggregation of 25 μm Aβ(1–42) in the absence (red) and in the presence of histidine-maltose PPI dendrimers. (Magenta) 25 μM Aβ(1–42) in the presence of dendrimers at dendrimer/peptide ratio = 0.1, (blue) 25 μM Aβ(1–42) in the presence of dendrimers at dendrimer/peptide ratio = 1. The temperature was 37°C, the pH was set to 7.4, and the concentration of ThT was 6 μM (adapted with permission from [22]).*

thus the interaction of dendrimer with other biomolecules is driven by hydrogen bonds, which is less strong; therefore, dendrimers covered by polysaccharides are less toxic [38, 39, 41].

In collaborations between the research groups of Dietmar Appelhans (Leibniz Institute of Polymer Research, Dresden, Germany), Josep Cladera (Autonomous University of Barcelona, Spain), and Isidro Ferrer in Barcelona (University of Barcelona, Spain), it has been shown that distinct PPI dendrimers with electroneutral maltose shell, with cationic maltose or maltotriose shell, were tested against amyloid toxicity in vivo and in vitro. The evaluation of the toxicity of Aβ in the presence of PPI maltose dendrimers showed that the dendrimers could significantly reduce the Aβ toxicity compared to Aβ alone [28].

Interestingly, only the electroneutral maltose dendrimers were able to reduce the toxicity of Alzheimer's disease brain extracts in cultured SH-SY5Y neuroblastoma cells [28]. Moreover, maltose PPI dendrimers with electroneutral or cationic surface penetrated the cytoplasm of cultured cells. Additionally, they penetrated inside the brain when administered to AD transgenic mice intranasally [28]. These PPI maltose dendrimers were able to modify amyloid plaque load in the brains of AD transgenic animals, showing anti-amyloid potential for in vivo applications. However, the studied maltose PPI dendrimers could not reverse memory impairment in APP/ PS1 mice following chronic administration. Strikingly, cationic maltose dendrimers were neurotoxic in vivo and caused cognitive decline in non-transgenic mice [28]. Taken together, these results suggest that maltose PPI dendrimers require further optimization of biocompatibility.

## **3. Modified PPI dendrimers as potential multifunctional therapeutics for Alzheimer's disease**

As it has been mentioned at the beginning of the chapter, Alzheimer's disease is a fatal neurodegenerative disorder. AD is characterized by a decade-long presymptomatic phase, and it is during the presymptomatic phase, before synaptic damage and neuronal loss, that therapies are most likely to be effective [43]. Thus, a preventive treatment which could protect synapses and reduce the neurotoxicity of Aβ

**95**

**Figure 5.**

*Glycodendrimers as Potential Multitalented Therapeutics in Alzheimer's Disease*

oligomers is one such strategy. Such successful drug candidates for AD treatment have to possess both anti-amyloidogenic and neuroprotective properties. Therefore,

To further improve the pharmacological properties of maltose PPI dendrimers, it was decided to modify PPI dendrimers of the fourth generation with maltose and histidine. Maltose was used due to anti-amyloidogenic properties; histidine was added due to several reasons: it is selectively transported through the BBB [44]. Histidine has chelating properties for Cu2+ ions [45]. Thus these properties were considered to be important since Cu ion dyshomeostasis may play a detrimental role in AD progression [46], and importantly, histidine has been shown to have some neuroprotective capacity [47]. After the modification, G4 PPI dendrimers modified with maltose and histidine were supposed to possess both anti-amyloid and neuro-

*Effect of G4 histidine-maltose PPI dendrimers on Aβ morphology. (A) Electron microscopy micrographs of 25 μM Aβ(1–40) incubated at pH 7.4 for 24 h. (B) 25 μM Aβ(1–40) incubated at pH 7.4 in the presence of G4 histidine-maltose PPI dendrimers at the ratio 1 to 1. (C) Aβ(1–42) incubated at pH 7.4 for 24 h. (D) Aβ(1–42) incubated in the presence of G4 histidine-maltose PPI dendrimers (clumped fibrils). Scale bar is 200 nm.*

a modification of maltose dendrimers with a molecule with neuroprotective characteristics was the next logical step in search of the new drug candidate for the

*DOI: http://dx.doi.org/10.5772/intechopen.88974*

protective properties simultaneously.

treatment of AD.

#### *Glycodendrimers as Potential Multitalented Therapeutics in Alzheimer's Disease DOI: http://dx.doi.org/10.5772/intechopen.88974*

oligomers is one such strategy. Such successful drug candidates for AD treatment have to possess both anti-amyloidogenic and neuroprotective properties. Therefore, a modification of maltose dendrimers with a molecule with neuroprotective characteristics was the next logical step in search of the new drug candidate for the treatment of AD.

To further improve the pharmacological properties of maltose PPI dendrimers, it was decided to modify PPI dendrimers of the fourth generation with maltose and histidine. Maltose was used due to anti-amyloidogenic properties; histidine was added due to several reasons: it is selectively transported through the BBB [44]. Histidine has chelating properties for Cu2+ ions [45]. Thus these properties were considered to be important since Cu ion dyshomeostasis may play a detrimental role in AD progression [46], and importantly, histidine has been shown to have some neuroprotective capacity [47]. After the modification, G4 PPI dendrimers modified with maltose and histidine were supposed to possess both anti-amyloid and neuroprotective properties simultaneously.

#### **Figure 5.**

*Neuroprotection - New Approaches and Prospects*

less toxic [38, 39, 41].

*with permission from [22]).*

**Figure 4.**

reduce the Aβ toxicity compared to Aβ alone [28].

optimization of biocompatibility.

**for Alzheimer's disease**

thus the interaction of dendrimer with other biomolecules is driven by hydrogen bonds, which is less strong; therefore, dendrimers covered by polysaccharides are

*Effect of G4 histidine-maltose PPI dendrimers on the fibrillization of Aβ. (A) Aggregation of 20 μM Aβ(1–40) in the absence (red) and the presence of histidine-maltose PPI dendrimers. (Magenta) 20 μM Aβ(1–40) in the presence of dendrimers at dendrimer/peptide ratio = 0.1, (blue) 20 μM Aβ(1–40) in the presence of dendrimers at dendrimer/peptide ratio = 1. (B) Aggregation of 25 μm Aβ(1–42) in the absence (red) and in the presence of histidine-maltose PPI dendrimers. (Magenta) 25 μM Aβ(1–42) in the presence of dendrimers at dendrimer/peptide ratio = 0.1, (blue) 25 μM Aβ(1–42) in the presence of dendrimers at dendrimer/peptide ratio = 1. The temperature was 37°C, the pH was set to 7.4, and the concentration of ThT was 6 μM (adapted* 

In collaborations between the research groups of Dietmar Appelhans (Leibniz Institute of Polymer Research, Dresden, Germany), Josep Cladera (Autonomous University of Barcelona, Spain), and Isidro Ferrer in Barcelona (University of Barcelona, Spain), it has been shown that distinct PPI dendrimers with electroneutral maltose shell, with cationic maltose or maltotriose shell, were tested against amyloid toxicity in vivo and in vitro. The evaluation of the toxicity of Aβ in the presence of PPI maltose dendrimers showed that the dendrimers could significantly

Interestingly, only the electroneutral maltose dendrimers were able to reduce the toxicity of Alzheimer's disease brain extracts in cultured SH-SY5Y neuroblastoma cells [28]. Moreover, maltose PPI dendrimers with electroneutral or cationic surface penetrated the cytoplasm of cultured cells. Additionally, they penetrated inside the brain when administered to AD transgenic mice intranasally [28]. These PPI maltose dendrimers were able to modify amyloid plaque load in the brains of AD transgenic animals, showing anti-amyloid potential for in vivo applications. However, the studied maltose PPI dendrimers could not reverse memory impairment in APP/ PS1 mice following chronic administration. Strikingly, cationic maltose dendrimers were neurotoxic in vivo and caused cognitive decline in non-transgenic mice [28]. Taken together, these results suggest that maltose PPI dendrimers require further

**3. Modified PPI dendrimers as potential multifunctional therapeutics** 

As it has been mentioned at the beginning of the chapter, Alzheimer's disease is a fatal neurodegenerative disorder. AD is characterized by a decade-long presymptomatic phase, and it is during the presymptomatic phase, before synaptic damage and neuronal loss, that therapies are most likely to be effective [43]. Thus, a preventive treatment which could protect synapses and reduce the neurotoxicity of Aβ

**94**

*Effect of G4 histidine-maltose PPI dendrimers on Aβ morphology. (A) Electron microscopy micrographs of 25 μM Aβ(1–40) incubated at pH 7.4 for 24 h. (B) 25 μM Aβ(1–40) incubated at pH 7.4 in the presence of G4 histidine-maltose PPI dendrimers at the ratio 1 to 1. (C) Aβ(1–42) incubated at pH 7.4 for 24 h. (D) Aβ(1–42) incubated in the presence of G4 histidine-maltose PPI dendrimers (clumped fibrils). Scale bar is 200 nm.*

In vitro evaluations demonstrated that histidine-maltose PPI dendrimers could interact with Aβ. As maltose PPI dendrimers, G4 histidine-maltose PPI dendrimers did not prevent fibril formation but clump Aβ fibrils (**Figure 5**). Importantly, small oligomeric aggregates were not present in the studied suspensions in the presence of the dendrimers. Interestingly, the intensity of ThT was significantly decreased following the aggregation of Aβ probably due to the competition of the dendrimers with ThT for binding to Aβ(1–40) or due to change of structure, resulting in lower ThT fluorescence quantum yield [48, 49]. To test if G4 histidine-maltose PPI dendrimers could reduce the neurotoxicity of Aβ, primary neurons derived from wildtype mouse were treated with 1 μM Aβ(1–42) in the presence of the dendrimers at the ratio 1 to 1. As it was demonstrated by cell viability assay, histidine-maltose PPI dendrimers significantly reduced the neurotoxicity of soluble Aβ oligomers [22]. **Figure 6** shows the neuronal viability in the presence of the dendrimers and Aβ(1–42) oligomers as assessed by a lactate dehydrogenase (LDH) activity assay. 1 μM G4 histidine-maltose PPI dendrimers were added to primary neurons and incubated 24 h before the assay; as it was documented, the dendrimers alone were not toxic to the neurons. 1 μM recombinant Aβ(1–42) monomers, oligomers, and fibrils were added to primary neurons and incubated 1 h at 37°C in the presence and the absence of dendrimers. The results demonstrate that G4 histidine-maltose PPI dendrimers significantly reduced the toxicity of Aβ(1–42) for primary neurons.

In vivo evaluations demonstrated that chronic treatment with histidine-maltose PPI dendrimers of APP/PS1 mice prevented AD-related memory impairment [22]. **Figure 7** shows the results of the memory test after the treatment. APP/PS1 mice harbor two human genes: APP with the KM670/671NL, the Swedish mutation, and PSEN1 with the L166P mutation [50]. In APP/PS1 mice, human Aβ increases with age, but Aβ42 is preferentially generated over Aβ40, and the expression of the human APP transgene is approximately 3-fold higher than the endogenous murine APP [51]. For the treatment, APP/PS1 and wild-type mice were randomly divided into four groups, two groups (transgenic and wild type) were treated intranasally with histidine-maltose PPI dendrimers, and two groups (transgenic and wild type) were given intranasally phosphate saline. Administration lasted 3 months until animals reached the age of 6 months, the age when the first cognitive decline is detected [52]. Memory evaluation tests were performed at the end of treatment using two object recognition tests in a VmazeR as described [52].

#### **Figure 6.**

*G4 histidine-maltose PPI dendrimers reduce the toxicity of Aβ oligomers for cultured primary neurons. (A) ThT fluorescence variation was used to monitor aggregation of 10 μM Aβ(1–42) in PBS at 37°C (black line); red line corresponds to ThT alone. The arrows indicate the time when aliquots of Aβ(1–42) were taken for neuronal viability assay. Aβ-M, a monomeric form of Aβ(1–42); Aβ-O, an oligomeric form of Aβ(1–42); Aβ-F, mature fibrils of Aβ(1–42); (B) 1 μM of G4 histidine-maltose PPI dendrimers were added to primary neurons and incubated 24 h before a cell viability assay. Cell viability was assessed by a lactate dehydrogenase activity assay. For the assay, 1 μM Aβ(1–42) of monomers, oligomers, and fibrils were added to wild-type primary neurons and incubated 1 h at 37°C. statistics: one-way ANOVA followed by Tukey's post hoc test; data are expressed as mean ± SD. Primary neurons were derived from the brains of wild-type mouse embryos and cultured for 19 days. The experiment was performed in triplicate, one embryo per replica (adapted with permission from [22]).*

**97**

**Figure 8.**

**Figure 7.**

*Glycodendrimers as Potential Multitalented Therapeutics in Alzheimer's Disease*

*G4 histidine-maltose PPI dendrimers can protect memory in vivo. Memory performance in the V-maze shows significant improvement after preventive treatment with histidine-maltose PPI dendrimers. Treatment procedure: at the age of 3 months, animals were randomly divided into four groups; two groups control and APP/PS1 mice were given intranasally 5 μL of PBS, and two groups received intranasally 5 μg/day of G4 histidine-maltose PPI dendrimers (dendrimers). Treatment lasted 3 months until animals reached the age of 6 months when APP/PS1 mice display cognitive impairment [52]. Statistics: two-way ANOVA with genotype and treatment as between factors followed by Tukey's post hoc test; data are expressed as mean ± SEM (adapted with permission from [22]).*

*G4 histidine-maltose PPI dendrimers protect synapses in vivo. (A) Synapse is a junction between two neurons, which consist of pre- and postsynaptic terminals characterized by specific pre- and postsynaptic proteins. Synaptophysin was used to assess presynapse, while drebrin was used to evaluate postsynapse. Brain tissue homogenates of control mice and mice treated with G4 histidine-maltose PPI dendrimers (dendrimers) were analyzed using Western blotting; β-actin was used for protein normalization. Statistics: Student's t-test (N is the number of animals per group, Western blotting was done in triplicate). Data are expressed as mean ± SD.*

*DOI: http://dx.doi.org/10.5772/intechopen.88974*

*Glycodendrimers as Potential Multitalented Therapeutics in Alzheimer's Disease DOI: http://dx.doi.org/10.5772/intechopen.88974*

#### **Figure 7.**

*Neuroprotection - New Approaches and Prospects*

using two object recognition tests in a VmazeR

In vitro evaluations demonstrated that histidine-maltose PPI dendrimers could interact with Aβ. As maltose PPI dendrimers, G4 histidine-maltose PPI dendrimers did not prevent fibril formation but clump Aβ fibrils (**Figure 5**). Importantly, small oligomeric aggregates were not present in the studied suspensions in the presence of the dendrimers. Interestingly, the intensity of ThT was significantly decreased following the aggregation of Aβ probably due to the competition of the dendrimers with ThT for binding to Aβ(1–40) or due to change of structure, resulting in lower ThT fluorescence quantum yield [48, 49]. To test if G4 histidine-maltose PPI dendrimers could reduce the neurotoxicity of Aβ, primary neurons derived from wildtype mouse were treated with 1 μM Aβ(1–42) in the presence of the dendrimers at the ratio 1 to 1. As it was demonstrated by cell viability assay, histidine-maltose PPI dendrimers significantly reduced the neurotoxicity of soluble Aβ oligomers [22]. **Figure 6** shows the neuronal viability in the presence of the dendrimers and Aβ(1–42) oligomers as assessed by a lactate dehydrogenase (LDH) activity assay. 1 μM G4 histidine-maltose PPI dendrimers were added to primary neurons and incubated 24 h before the assay; as it was documented, the dendrimers alone were not toxic to the neurons. 1 μM recombinant Aβ(1–42) monomers, oligomers, and fibrils were added to primary neurons and incubated 1 h at 37°C in the presence and the absence of dendrimers. The results demonstrate that G4 histidine-maltose PPI dendrimers significantly reduced the toxicity of Aβ(1–42) for primary neurons. In vivo evaluations demonstrated that chronic treatment with histidine-maltose PPI dendrimers of APP/PS1 mice prevented AD-related memory impairment [22]. **Figure 7** shows the results of the memory test after the treatment. APP/PS1 mice harbor two human genes: APP with the KM670/671NL, the Swedish mutation, and PSEN1 with the L166P mutation [50]. In APP/PS1 mice, human Aβ increases with age, but Aβ42 is preferentially generated over Aβ40, and the expression of the human APP transgene is approximately 3-fold higher than the endogenous murine APP [51]. For the treatment, APP/PS1 and wild-type mice were randomly divided into four groups, two groups (transgenic and wild type) were treated intranasally with histidine-maltose PPI dendrimers, and two groups (transgenic and wild type) were given intranasally phosphate saline. Administration lasted 3 months until animals reached the age of 6 months, the age when the first cognitive decline is detected [52]. Memory evaluation tests were performed at the end of treatment

as described [52].

*G4 histidine-maltose PPI dendrimers reduce the toxicity of Aβ oligomers for cultured primary neurons. (A) ThT fluorescence variation was used to monitor aggregation of 10 μM Aβ(1–42) in PBS at 37°C (black line); red line corresponds to ThT alone. The arrows indicate the time when aliquots of Aβ(1–42) were taken for neuronal viability assay. Aβ-M, a monomeric form of Aβ(1–42); Aβ-O, an oligomeric form of Aβ(1–42); Aβ-F, mature fibrils of Aβ(1–42); (B) 1 μM of G4 histidine-maltose PPI dendrimers were added to primary neurons and incubated 24 h before a cell viability assay. Cell viability was assessed by a lactate dehydrogenase activity assay. For the assay, 1 μM Aβ(1–42) of monomers, oligomers, and fibrils were added to wild-type primary neurons and incubated 1 h at 37°C. statistics: one-way ANOVA followed by Tukey's post hoc test; data are expressed as mean ± SD. Primary neurons were derived from the brains of wild-type mouse embryos and cultured for 19 days. The experiment was performed in triplicate, one embryo per replica (adapted with permission from [22]).*

**96**

**Figure 6.**

*G4 histidine-maltose PPI dendrimers can protect memory in vivo. Memory performance in the V-maze shows significant improvement after preventive treatment with histidine-maltose PPI dendrimers. Treatment procedure: at the age of 3 months, animals were randomly divided into four groups; two groups control and APP/PS1 mice were given intranasally 5 μL of PBS, and two groups received intranasally 5 μg/day of G4 histidine-maltose PPI dendrimers (dendrimers). Treatment lasted 3 months until animals reached the age of 6 months when APP/PS1 mice display cognitive impairment [52]. Statistics: two-way ANOVA with genotype and treatment as between factors followed by Tukey's post hoc test; data are expressed as mean ± SEM (adapted with permission from [22]).*

#### **Figure 8.**

*G4 histidine-maltose PPI dendrimers protect synapses in vivo. (A) Synapse is a junction between two neurons, which consist of pre- and postsynaptic terminals characterized by specific pre- and postsynaptic proteins. Synaptophysin was used to assess presynapse, while drebrin was used to evaluate postsynapse. Brain tissue homogenates of control mice and mice treated with G4 histidine-maltose PPI dendrimers (dendrimers) were analyzed using Western blotting; β-actin was used for protein normalization. Statistics: Student's t-test (N is the number of animals per group, Western blotting was done in triplicate). Data are expressed as mean ± SD.*

To understand a possible mechanism behind the memory rescue, the levels of pre- and postsynaptic markers in the brain of treated APP/PS1 mice were evaluated by Western blotting. Pre- and postsynaptic markers, such as drebrin and synaptophysin, play a crucial role in the synaptic plasticity and are downregulated in AD [53, 54]. Loss of synaptophysin correlates with cognitive impairments in AD patients and AD transgenic models [54, 55]; Psd95 knockout animals have impaired basal synaptic transmission and learning deficit [56]; transgenic animals lacking synaptophysin have reduced novel object recognition [57]. Importantly, it has been shown that loss of synaptophysin immunoreactivity precedes amyloid plaque formation [58, 59]. Preventive treatment of AD transgenic mice with G4 histidinemaltose PPI dendrimers prevented a decrease in synaptic proteins compared to PBS-treated mice [22].

In contrast, G4 histidine-maltose PPI dendrimers did not change the level of these synaptic proteins in WT mice, indicating that, most likely, the level of their mRNA expression was not affected [22]. Thus it is reasonable to think that the increased levels of pre- and postsynaptic proteins are more likely an effect of reduced synaptic loss in the treated AD transgenic animals (**Figure 8**). Thus a possible mechanism of memory protection in APP/PS1 could be the synapses were shielded by the dendrimers from toxic Aβ oligomers or the toxicity of Aβ oligomers were inactivated in the presence of the dendrimers.

#### **4. Conclusions and perspectives**

Dendrimers, which represent a type of 3D polymers, have been in the spotlight for three decades in biomedical and pharmaceutical research, and their chemistry and synthesis are continuously progressing by efforts from many research groups and companies. Although there are still many unclear problems in AD, in this chapter, functionalization of dendrimers dedicated to the prevention of memory decline in AD pathogenesis has been discussed. Based on the reviewed literature, PPI dendrimers have been shown to be useful in the way of the surface functionalization, which tuned their biochemical properties. Strikingly, the effect of the surface functionalization with histidine and maltose magnified exponentially neuroprotective properties of PPI dendrimers, resulting in an unprecedented outcome, such as memory protection in AD transgenic animals.

In this chapter, I have analyzed the functionalization of PPI dendrimers, which tuned the intrinsic properties of PPI dendrimers and converted them into a multifunctional drug candidate against Alzheimer's disease. Modification of the dendrimer surface with maltose allowed dendrimers successfully to interfere with Aβ(1–42) by forming nontoxic hybrid glycofibrils. Modification of the dendrimer surface with histidine improved the ability of the dendrimers to cross the blood– brain barrier and resulted in synaptic protection. By reducing the level of soluble amyloid oligomers, on the one hand, and conferring synapse protection, on the other hand, the dendrimers were given multifunctionality against main features of AD, synaptic loss, and aggregation of Aβ. These observations, coming out of the studies on the interaction of dendrimers with amyloid peptides [18, 22, 28, 32, 42], carried out in vitro and in vivo, point toward a possible use of dendrimers (in particular functionalization of PPI dendrimers with histidine and maltose) as a multifunctional drug candidate against Alzheimer's disease.

However, to find a successful drug against AD, other modifications of histidinemaltose PPI dendrimers might be required. For example, the ability to cross the blood-brain barrier, cell wall penetration, distribution in the specific tissue, and biodegradation could be tuned for a particular dendrimer application.

**99**

**Author details**

Oxana Klementieva

University, Lund, Sweden

provided the original work is properly cited.

*Glycodendrimers as Potential Multitalented Therapeutics in Alzheimer's Disease*

I acknowledge Dr. Dietmar Appelhans (Leibniz Institute of Polymer Research Dresden, Dresden, Germany) for the generation of histidine-maltose PPI den-

The work is supported by MultiPark (Lund University), Vinnova, Swedish

I thank Dr. Stefan Broselid for fruitful discussions and editorial help.

Department of Experimental Medical Science, Faculty of Medicine, Lund

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: oxana.klementieva@med.lu.se

*DOI: http://dx.doi.org/10.5772/intechopen.88974*

drimers, collaboration, and valuable discussions.

**Acknowledgements**

Research Council grants.

**Thanks**

*Glycodendrimers as Potential Multitalented Therapeutics in Alzheimer's Disease DOI: http://dx.doi.org/10.5772/intechopen.88974*
